The Leiden Malaria Research group contributed to a study showing that malaria parasites can survive and develop in dendritic cells (published in PNAS, June 2011)
In this study it has been shown that blood stages of the rodent malaria parasite can survive and develop in CD317(+) dendritic cells. Small numbers of these cells released parasites that were infectious for erythrocytes in vivo.
The Leiden Malaria Research Group and collaborators showed that the RNA binding protein pumilio (puf2) is a key regulator of the transformation of malaria parasites during transmission of the sporozoite from the mosquito to the liver of the mammalian host (published in Plos Pathogens)
The Leiden Malaria Research Group developed the piggyBac transposable system for random mutagenesis in the malaria parasite Plasmodium berghei (published in BMC Genomics).
Transposable elements have been widely used to induce insertional mutagenesis in highly diverse biological systems. The TTAA-specific transposon piggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species, particularly insects. We have developed the piggyBac transposable system for random mutagenesis in the malaria parasite Plasmodium berghei. See also the commentary in BMC Biology.
The Leiden Malaria Research Group published a review on sequestration of malaria infected red blood cells (PloS Pathogens)
The sequestration of Plasmodium falciparum-infected red blood cells (irbcs) in the microvasculature of organs is associated with severe disease; correspondingly, the molecular basis of irbc adherence is an active area of study. In contrast to the human parasite P. falciparum, much less is known about sequestration in other malaria parasites, including those species that are used as models to study severe malaria. Here, we review the cytoadherence properties of irbcs of the rodent parasite Plasmodium berghei ANKA, where schizonts demonstrate a clear sequestration phenotype. The role of sequestration is discussed in the context of disease as are the general (dis)similarities of P. berghei and P. falciparum sequestration.
The Leiden Malaria Research Group and collaborators developed a method for removal of heterologous sequences from the genome of malaria parasites using FLPe-recombinase (published in PloS One).
Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. We developed of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines.
The Leiden Malaria Research group contributed to a study showing that malaria parasites (sporozoites) can develop in the skin (PNAS, Oct 2010)
The first step of malaria parasite (Plasmodium) development in vertebrate host is the transformation of the sporozoite, the parasite stage injected by the mosquito in the skin, into merozoites, the stage that invades erythrocytes and initiates the disease. The current view is that, in mammals, this stage conversion occurs only inside hepatocytes. In this study it is shown the transformation of sporozoites of rodent-infecting malaria parasites into merozoites in the skin of mice. Therefore, during malaria in rodents, the skin is not just the route to the liver but is also the final destination for many inoculated parasites, where they can differentiate into merozoites and possibly persist. It has to be established if the same developmental processes also occur in human-infecting malaria parasites, but if they do, this will have major implications on what sort of immune responses will be generated against the parasite.
The Leiden Malaria Research group published a paper describing the (use of the) RMgm-database (Trends Parasitol. July 2010).
The RMgm database, www.pberghei.eu, is a web-based, manually curated, repository containing information on the genotype and phenotype of genetically modified rodent-malaria parasites generated by many labs worldwide. In addition it contains information on gene function, inferred from mutant phenotypes.
In the paper special emphasis has been placed on standardization of generating and describing mutant genotypes. See the table (PDF-file) containing suggestions for standardization of generating and describing rodent parasite mutants. In this paper the use of standardized vocabularies to describe mutant phenotypes and the use of Gene Ontology (GO) for annotating gene function of Plasmodium genes is discussed.
The Leiden Malaria Research Group and collaborators analysed the enzyme glutathione reductase involved in glutathione (GSH) metabolism of malaria parasites.
It was shown that glutathione reductase (GR) is essential for the development of the malaria parasite in mosquitoes. In contrast, GR is not essential for growth and multiplication of the blood stages of this parasite. These studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism. For example, it was shown that GR-null parasites had the same sensitivity to methylene blue and Eosin B as wild type parasites demonstrating that these compounds target molecules other than GR in malaria parasites (see article in Journal of Biological Chemistry)
The Leiden Malaria Research Group and collaborators identified malaria proteins that play a role in Plasmodium gamete fertility and fertilisation.
These proteins belong to a family of 10 structurally related proteins, the so called 6-cys family (see the article in PloS Pathogens).
The Leiden Malaria Research Group and collaborators generated a Plasmodium Artificial Chromosome, which can be stably segregated and maintained in parasites.
The artificial chromosome represents a useful tool for gene transfer, both as cloning vectors and in chromosome biology research (see article in CELL, Host & Microbe , Cicero and news item LUMC website)
The Leiden Malaria Research Group and collaborators identified proteins associated with a messenger ribonucleoprotein particle (mRNP) that is involved in translational repression in malaria parasites.
Plasmodium female gametes contain mRNPs that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from P. berghei gametocytes defined by a RNA helicase (DOZI) and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. This study defines Plasmodium P-granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection (see article in PloS Pathogens).
The Leiden Malaria Research Group and collaborators generated virulence attenuated malaria parasites that induce protective immunity against experimental malaria
This study demonstrates, for the first time, that a single gene disruption in the rodent malaria parasite P. berghei can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines (see article in Am. J. Pathol.).
The Leiden Malaria Research Group and collaborators developed a method for visualisation and quantitative analysis of malaria liver stages by real time imaging.
A transgenic malaria parasite of rodents, P. berghei, expressing the bioluminescent reporter protein luciferase, is used to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium. (see article in PloS One)
A Grand Challenges Exploration (GCE) Grant, funded by The Gates Foundation, awarded to the LMRG
Shahid Khan, one of the researchers of the Leiden Malaria Research Group, has been awarded a $100,000 grant from the Gates Foundation for exploratory research on malaria. His project was one of 76 selected from almost 3,000 proposals. To date 262 researchers representing 30 countries have been awarded these grants. Khan’s winning project is entitled: ‘Preventing Malaria in Both Host and Vector’ and the aim of this project is to use genetically attenuated parasites as vehicles to not only protect the host but also kill the parasite in the mosquito. The grant covers research costs for 1 year, up to $100.000, which Khan’s team (part of the Leiden Malaria Research Group (LUMC); headed by Chris Janse) will use to perform this exploratory research. See also the news items on the LUMC homepage and the TI Pharma website.
The Leiden Malaria Group contributed to a study describing a cyclic GMP signalling module that regulates gliding motility of Plasmodium ookinetes, published in PloS Pathogens
Through genetic interaction a signalling module is described that identifies guanosine 3′, 5′-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase δ (PDEδ), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase β (GCβ), showing that in ookinetes GCβ is an important source for cGMP, and that PDEδ is the relevant cGMP degrading enzyme.
Member of Parasitology wins prize for 'outstanding' presentation
Aga Religa (PhD student) from the Leiden Malaria Research Group (Parasitology) was awarded a prize at the 2009 annual Molecular Parasitology Meeting, Woods Hole (MA, USA), for her ‘outstanding’ presentation of her research on the role of a histone deacetylase (SIR2) in malaria parasites. She found that the SIR2 protein, which is involved in regulation of chromatin structure and gene transcription, also plays an unexpected role in the parasites development inside the mosquito (see also the abstract of the presentation). The PhD project is a joint collaboration between Leiden Malaria Leiden Group at the LUMC (Chris Janse), the University of Glasgow (Andy Waters) and the Pasteur Institute, Paris (Artur Scherf). The prize consists of funding to attend and to present her work at the annual meeting of the American Society of Tropical Medicine and Hygiene in Washington DC (USA) in November 2009 as well as a one year subscription to the Journal Cellular Microbiology. See also the publication on the LUMC Albinusnet and an abstract of her research.
Links between the malaria database PlasmoDB and the Leiden RMgm database:
In the new release of the malaria database PlasmoDB (www.plasmodb.org) there are direct links from appropriate gene pages to genetically modified rodent malaria parasites in the RMgm database (www.pberghei.eu) of the Leiden Malaria Research Group. In PlasmoDB see the ‘external links – Databases’: Phenotypes from Genetically Modified Rodent Malaria Parasite database. By clicking on this link, a list is shown of all mutants in the RMgm database which relate to the gene (e.g. gene has been disrupted, tagged, mutated, expressed as a transgene etc). Clicking on a specific mutant directs to the RMgm webpage and here one can find the more detailed information on the mutant. In the attachment you will find more information on the RMgm database (i.e. ‘What is the RMgm database’; ‘What the RMgm database offers’; ‘How you can contribute to the database’; ‘Preferred information (requirements) for a RMgm mutant entry’; etc).
Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites
RNA interference (RNAi) is an evolutionarily conserved mechanism found across a range of eukaryotes, where it plays a key role in post-transcriptional gene regulation. With its exquisite specificity for the target gene as well as its potent and reversible action, RNAi technology has now become a standard technique in the molecular toolbox for reverse genetic experimentation in many systems, providing a quick and easy means to gain valuable insight into gene function, in particular those that are essential to cell viability. The use of RNA interference (RNAi) to silence gene expression of malaria parasites would provide a powerful means to gain valuable insight in the function of genes that are essential for growth of the patahogenic blood stages.
The Leiden malaria Group and collaborators provide strong evidence that RNAi is absent in malaria parasites and therefore it is unlikely that RNAi-based gene silencing will prove to be a reliable approach to unravel the function of malaria proteins. See for more information the publication in Nucleic Acids Research.
Updated and new information on our SharePoint website (containing the LMRG P. berghei protocols and databases).
Specifically: 1) The effect of host (animal) diet upon rodent malaria parasite infections. We have received several of enquiries about host diet and P. berghei infections. In our experience host diets can influence both the course of an infection and its virulence phenotype (and also on the quality of parasites for transfection). We have provided some additional information on host diet and its influence on rodent malaria parasite infections (see FAQ and the folder Shared Documents, subfolder Host diet). Since P. berghei is frequently (and increasingly) used to study host-parasite interactions we believe standardization of diets in experiments (and between labs) may be very useful. 2) Problems using one of LMRG a standard DNA vectors (pL0037) for genetic modification: unwanted integration. We have on many occasions successfully used our positive/negative selection vectors, pl0035 (available via MR4) and its close derivative pL0037, to target P. berghei genes by gene replacement and then using cloned lines of these mutant parasites we have been able to use negative selection (i.e. 5-FC) to ‘re-cycle’ and remove the drug selectable marker cassette. Recently, however, we have noticed that a number of our transfections using pL0037 appear not to be integrating into the correct locus. For more information see NEWS item on our Sharepoint site.
Login details (username, password) for this Sharepoint-site can be requested by filling out the ‘request for access/password’ form on our website
The Leiden Malaria Group contributed to a study of the glutathione biosynthetic pathway, published in PloS Pathogens
Infection of red blood cells subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Malaria parasites have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. This study demonstrates that gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, is not essential for the growth of the pathogenic blood stages but is essential for development of the parasite in the mosquito.
These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito
The Leiden Malaria Group contributed to a study of fatty acid synthesis, published in CELL host & microbe
The Leiden Group contributed to a paper on fatty acid synthesis in malaria parasites. The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in malaria infections. Synthetic chemistry and transfection studies demonstrate that the FabI enzyme of Plasmodium is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. The lack of of fabI does not impede blood-stage growth. In contrast, the growth of the liver stafes of the parasite is markedly affected
These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.
The Leiden Malaria Research Group has a new website:
The Leiden Malaria Research Group has set up a publically accessible database Pberghei.eu.
Pberghei.eu hosts a database containing information on genetically modified rodent malaria parasite lines generated by many labs worldwide.
Specifically, we are collating data that has been generated in the three rodent parasite species; Plasmodium berghei, P. yoelii and P. chabaudi. The aim of this database is to provide the research community access to detailed information on the generation (e.g. disruption, tagging, mutation, transgene expression) and phenotype (e.g. lethal, aberrant growth, failure of sporozoites to infect liver cells, expression of reporter genes) of genetically modified malaria parasites.
We are still technically improving the database and are in the process of adding in many more genetically modified parasite lines (and their associated information). At this moment we have information on around 100 parasite lines. We will not only provide information on ‘successful’ mutants with a clear phenotype, but also (un)published mutants without a clear phenotype and (un)published negative trials to disrupt genes.
Currently we are in discussion with PlasmoDB on the best way to link our RMGM database with theirs to best ensure availability and access of the collated information.
We would be pleased and would very much welcome everyone’s comments to improve our RMGM database. Moreover, we would like to explore the possibilities of coordinating our activities with you and other researchers; such that individuals can submit and/or upload information on their rodent parasite mutants either directly onto the RMGM database or in an excel format that can be sent to the Leiden Malaria Research Group for uploading. We believe that the information on published and unpublished (rodent) malaria parasite mutants, all within one database, will help the research of all and may also prevent unnecessary duplication of work/experiments.