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Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

Malaria control relies on passive case detection, and this strategy fails detecting asymptomatic infections. In addition, infections in endemic areas harbor multiple parasite genotypes that could affect case management and malaria epidemiology. Here, we performed AmpSeq genotyping to capture polymorphisms associated with antimalarial resistance and the genetic diversity within natural Plasmodium falciparum infections. Known genetic polymorphisms associated with altered drug susceptibility were screened for the five most common marker genes, pfdhfr, pfdhps, pfmdr1, pfcrt, and pfK13, and genetic diversity was established from two known AmpSeq markers, cpmp and csp. Relative abundance of the different genotypes within mixed infections was calculated from the number of reads per genotype. Genotyping was performed on 117 samples, 63 from asymptomatic and 54 from symptomatic individuals. We identified up to 15 genotypes within an infection, and the median multiplicity of infection was higher in asymptomatic infections (median MOI = 5 in asymptomatics versus median MOI = 2 in symptomatics, P

Experimental studies on the biology of malaria parasites have been mostly based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of somePlasmodium falciparumclinical isolates, in analyses that focused on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points during several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and timepoints. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity although proportions of genotypes varied over time. Drug-resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (includingAP2-HS, EPACandSRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identifiedde novovariants not detected in the bulk isolate sequences. Interestingly, most of these were nonsense mutants and frameshifts disrupting the coding sequence ofEPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of Identity-By-Descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within parasite populations.

Background In areas where Plasmodium falciparum malaria is seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season. Methods In The Gambia, a cohort of 42 individuals with quantitative polymerase chain reaction-positive P falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate infection persistence. The influence of human host and parasitological factors was investigated. Results A large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16 of 40). At the start of the dry season, the majority of these persistent infections (82%) had parasite densities >10 p/µL compared to only 5.9% of short-lived infections. Persistent infections (59%) were also more likely to be multiclonal than short-lived infections (5.9%) and were associated with individuals having higher levels of P falciparum-specific antibodies (P = .02). Conclusions Asymptomatic persistent infections were multiclonal with higher parasite densities at the beginning of the dry season. Screening and treating asymptomatic infections during the dry season may reduce the human reservoir of malaria responsible for initiating transmission in the wet season.

BackgroundIn areas where Plasmodium falciparum malaria is highly seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season, bridging transmission seasons several months apart. Understanding infections during the dry season could thus inform approaches for malaria control.MethodsIn The Gambia, a cohort of 42 individuals with qPCR positive P. falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate the duration of detectable infections. The influence of human host (age, sex, haemoglobin concentration and genotype, and P. falciparum-specific antibodies), and parasitological (parasite density, gametocyte density and genotypic multiplicity of infection) factors was investigated.ResultsA large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16/40), with the majority of these infections also harbouring gametocytes (81.3%; 13/16). 22 infections were classified as persistent (detectable for at least 3 months), 17 were classified as short-lived (undetectable within 2 months), and 3 were treated (due to symptoms). At the start of the dry season, the majority of persistent infections (82%; 18/22) had parasite densities >10 p/µL compared to only 5.9% (1/17) of short-lived infections. Persistent infections (59%; 13/22) were also more likely to be multi-clonal than short-lived infections (5.9%; 1/17), they were most common in 5 to 15 year old children (63%; 12/19), and were associated with individuals having higher levels of P. falciparum-specific antibodies (p = 0.058).ConclusionsAsymptomatic persistent dry season infections in The Gambia were multiclonal with higher parasite densities at the beginning of the dry season, mostly occurring in school age children and adults with higher P. falciparum-specific antibodies. Screening and treating asymptomatic, malaria-infected individuals during the dry season may reduce the human reservoir of malaria responsible initiating transmission in the wet-season.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization., Author summary Globalization has led to the spread of pathogens through increased human movement. Microbiologists track epidemics of these pathogens by cataloguing geographic diversity in the genes that encode for variant surface antigens (VSA). Here, we developed a computational approach to explore the evolution of specific DNA sequences of the major VSA gene of the human malaria parasite, Plasmodium falciparum. First, we tested the method by comparing DNA sequences of these genes from P. falciparum to those of Plasmodium species that infect chimpanzees and gorillas. We showed that it could distinguish DNA signatures specific to each species. Next, we asked whether our method could detect geographic signatures within these genes by analyzing a global collection of P. falciparum isolates from 23 locations in 10 countries. The important outcome of our work was the ability to identify geographic signatures specific to countries and continents that were consistent with the “out of Africa” origin of P. falciparum. We can now identify malaria parasites from countries within Africa, South America, and Asia/Oceania using a diverse region of VSA genes without having to sequence and assemble whole parasite genomes. This methodology has potential applications in malaria surveillance to track parasites as they move around the world.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

International audience; The last malaria parasite standing will display effective adaptations to selective forces. While substantial progress has been made in reducing malaria mortality, eradication will require elimination of all Plasmodium parasites, including those in asymptomatic infections. These typically chronic, low-density infections are difficult to detect, yet can persist for months. We argue that asymptomatic infection is the parasite’s best asset for survival but it can be exploited if studied as a new model for host–pathogen–vector interactions. Regular sampling from cohorts of asymptomatic individuals can provide a means to investigate continuous parasite development within its natural host. State-of-the-art techniques can now be applied to such infections. This approach may reveal key molecular drivers of chronic infections – a critical step for malaria eradication.

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence., Lindsay Stewart et al. analyze clinical isolates of the human malaria parasite Plasmodium falciparum from a highly endemic West African population and show that intrinsic multiplication rate variation is associated with blood-stage infection intensity. Their results indicate that parasite control of multiplication contributes to virulence.

Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters. Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates. We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

International audience; Background. In Plasmodium falciparum-infected patients treated with artemisinins, parasitemia declines through so-called pitting, an innate splenic process that transforms infected red blood cells (iRBCs) into once-infected RBCs (O-iRBCs).Methods. We measured pitting in 83 French travelers and 42 Malian children treated for malaria with artesunate.Results. In travelers, O-iRBCs peaked at 107.7% initial parasitemia. In Malian children aged 1.5-4 years, O-iRBCs peaked at higher concentrations than in children aged 9-13 years (91.60% vs 31.95%; P = .0097). The parasite clearance time in older children was shorter than in younger children (P = .0001), and the decline in parasitemia in children aged 1.5-4 years often started 6 hours after treatment initiation, a lag phase generally absent in infants and older children. A 6-hour lag phase in artificial pitting of artesunate-exposed iRBCs was also observed in vitro. The proportion of iRBCs recognized by autologous immunoglobulin G (IgG) correlated with the parasite clearance time (r = −0.501; P = .0006) and peak O-iRBC concentration (r = −0.420; P = .0033).Conclusions. Antimalarial immunity correlates with fast artemisinin-induced parasite clearance and low pitting rates. In nonimmune populations, artemisinin-induced P. falciparum clearance is related to pitting and starts after a 6-hour lag phase. In immune populations, passively and naturally acquired immune mechanisms operating faster than pitting may exist. This mechanism may mitigate the emergence of artemisinin-resistant P. falciparum in Africa.

Pathogen genome sequencing directly from clinical samples is quickly gaining importance in genetic and medical research studies. However, low DNA yield from blood-borne pathogens is often a limiting factor. The problem worsens in extremely base-biased genomes such as the AT-rich Plasmodium falciparum. We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing. We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome. We show that this method reduces amplification bias and chimera formation. Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.

The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7–72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle., Author Summary Malaria kills >600,000 people each year, with most deaths caused by Plasmodium falciparum. A family of proteins known as P. falciparum erythrocyte membrane protein 1, PfEMP1, is expressed on the surface of infected erythrocytes and plays an important role in pathogenesis. Each P. falciparum genome contains approximately 60 highly polymorphic var genes encoding the PfEMP1 proteins, and monoallelic expression with periodic switching results in immune evasion. Var gene polymorphism is thus critical to this survival strategy. We investigated how var gene diversity is generated by performing an in vitro evolution experiment, tracking var gene mutation in ‘real-time’ with whole genome sequencing. We found that genome structural variation is focused in and around var genes. These genetic rearrangements created new ‘chimeric’ var gene sequences during the mitotic part of the life cycle, and were consistent with processes of mitotic non-allelic homologous recombination. The recombinant var genes were always in frame and with conserved overall var gene architecture, and the recombination rate implies that many millions of rearranged var gene sequences are produced every 48-hour life cycle within infected individuals. In conclusion, we provide a detailed description of how new var gene sequences are continuously generated in the parasite genome, helping to explain long-term parasite survival within infected human hosts.

For reasons that remain unknown, the Plasmodium falciparum genome has an exceptionally high AT content compared to other Plasmodium species and eukaryotes in general - nearly 80% in coding regions and approaching 90% in non-coding regions. Here, we examine how this phenomenon relates to genome-wide patterns of de novo mutation. Mutation accumulation experiments were performed by sequential cloning of six P. falciparum isolates growing in human erythrocytes in vitro for 4 years, with 279 clones sampled for whole genome sequencing at different time points. Genome sequence analysis of these samples revealed a significant excess of G:C to A:T transitions compared to other types of nucleotide substitution, which would naturally cause AT content to equilibrate close to the level seen across the P. falciparum reference genome (80.6% AT). These data also uncover an extremely high rate of small indel mutation relative to other species, primarily associated with repetitive AT-rich sequences, in addition to larger-scale structural rearrangements focused in antigen-coding var genes. In conclusion, high AT content in P. falciparum is driven by a systematic mutational bias and ultimately leads to an unusual level of microstructural plasticity, raising the question of whether this contributes to adaptive evolution.

Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum . The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes ( HB3var3 , 3D7_PFD0020c , ITvar7 , and ITvar19 ) that showed 11- to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann–Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann–Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (, Author Summary Malaria remains one of the world's most deadly diseases. Life-threatening malaria is linked to a process called rosetting, in which malaria parasite-infected red blood cells bind to uninfected red cells to form aggregates that block blood flow in vital organs such as the brain. Current efforts to develop drugs or vaccines against rosetting are hindered by variation in the parasite rosette-mediating proteins, found on the surface of infected red cells. We studied these parasite-derived surface proteins and discovered that although they are variable, they share some common features. We raised antibodies against the rosette-mediating proteins, and found that they cross-reacted with multiple rosetting parasite strains from different countries around the world, including samples collected directly from African children with severe malaria. These findings provide new insights into malaria parasite interactions with human cells, and provide proof of principle that variable parasite molecules from virulent malaria parasites can induce strain-transcending antibodies. Hence, this work provides the foundation for the development of new therapies to treat or prevent life-threatening malaria.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain(2-4). This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in (5)). Sequestration is also found in most microvascular tissues of the human body(2, 3). The mechanism by which iRBC attach to the blood vessel walls is still poorly understood. The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier(6). However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo. A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype. In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs(7).

Background The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes for each VSA gene. Methods All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer software. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray. Results From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. Bioinformatic analysis of the new VSA sequences showed that 13 rif genes and five stevor genes were well-conserved across at least three strains (83-100% amino acid identity). The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly up-regulated gene in rosetting parasites was ITvar9 (19 to 429-fold up-regulated over six time points). Only one rif gene (IT4rifA_042) was up-regulated by more than four fold (five fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were up-regulated by more than two fold. 377 non-VSA genes were differentially expressed by three fold or more in rosetting parasites, although none was as markedly or consistently up-regulated as ITvar9. Conclusions Probes for the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation.

Cerebral malaria is characterised by an accumulation of infected erythrocytes in the microvasculature of the brain. P. falciparum infected erythrocytes have been shown to bind to a Human Brain Endothelial Cell line (HBEC-5i), however, the parasite adhesion ligands necessary to anchor the infected erythrocytes onto HBEC-5i have not been identified. We aimed to identify parasite variant surface antigens that are differentially transcribed after selection for cytoadherence to HBEC-5i. The P. falciparum laboratory strains 3D7, HB3 and IT/FCR3 were selected using a panning assay for binding to HBEC-5i. In order to analyse their transcriptome using a microarray chip based on the 3D7 genome, variant surface antigens sequences (var/PfEMP1, rif, stevor) were extracted from the sequenced HB3 and IT genomes and added to the 3D7-based microarray chip. Microarray analysis was carried out on selected and unselected parasites of all three strains. Transcriptome data clearly indicated that the most highly upregulated genes after selection were group A or group A-like var genes, showing 11 to over 100 fold upregulation in selected parasites. The rif gene located head-to-head to the upregulated var gene was also highly expressed. To a lesser extent some exported proteins like RESA-1, PfEMP3 or PHIST family members also showed increased transcription in HBEC-selected parasites (2-3 fold upregulation). Reverse transcriptase-PCR confirmed the upregulation of group A var genes in selected parasites, suggesting that the group A PfEMP1 variants are major candidate ligands for parasite binding to HBEC5i. These findings are consistent with previous work showing an association between group A var genes and cerebral malaria. The HBEC-5i receptors mediating this interaction with infected erythrocytes are currently under investigation. Author details IIIR, Ashworth Laboratories, University of Edinburgh, Edinburgh, EH9 3JT, UK. Nanyang Technological University, SBS, 60 Nanyang Drive, 637551, Singapore.

Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

Severe malaria has a high mortality rate (15–20%) despite treatment with effective antimalarial drugs. Adjunctive therapies for severe malaria that target the underlying disease process are therefore urgently required. Adhesion of erythrocytes infected withPlasmodium falciparumto human cells has a key role in the pathogenesis of life-threatening malaria and could be targeted with antiadhesion therapy. Parasite adhesion interactions include binding to endothelial cells (cytoadherence), rosetting with uninfected erythrocytes and platelet-mediated clumping of infected erythrocytes. Recent research has started to define the molecular mechanisms of parasite adhesion, and antiadhesion therapies are being explored. However, many fundamental questions regarding the role of parasite adhesion in severe malaria remain unanswered. There is strong evidence that rosetting contributes to severe malaria in sub-Saharan Africa; however, the identity of other parasite adhesion phenotypes that are implicated in disease pathogenesis remains unclear. In addition, the possibility of geographic variation in adhesion phenotypes causing severe malaria, linked to differences in malaria transmission levels and host immunity, has been neglected. Further research is needed to realise the untapped potential of antiadhesion adjunctive therapies, which could revolutionise the treatment of severe malaria and reduce the high mortality rate of the disease.

International audience; Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at syn-apses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/ NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.

Erratum to previous paper on schizophrenia related proteins "DISC1, PDE4B, and NDE1 at the centrosome and synapse"