The human malaria parasite Plasmodium falciparum can sense environmental changes and respond by antigenic switching.

The primary antigenic and virulence determinant of the human malaria parasite Plasmodium falciparum is a variant surface protein called PfEMP1. Different forms of PfEMP1 are encoded by a multicopy gene family called var, and switching between active genes enables the parasites to evade the antibody response of their human hosts. var gene switching is key for the maintenance of chronic infections; however, what controls switching is unknown, although it has been suggested to occur at a constant frequency with little or no environmental influence. var gene transcription is controlled epigenetically through the activity of histone methyltransferases (HMTs). Studies in model systems have shown that metabolism and epigenetic control of gene expression are linked through the availability of intracellular S-adenosylmethionine (SAM), the principal methyl donor in biological methylation modifications, which can fluctuate based on nutrient availability. To determine whether environmental conditions and changes in metabolism can influence var gene expression, P. falciparum was cultured in media with altered concentrations of nutrients involved in SAM metabolism. We found that conditions that influence lipid metabolism induce var gene switching, indicating that parasites can respond to changes in their environment by altering var gene expression patterns. Genetic modifications that directly modified expression of the enzymes that control SAM levels similarly led to profound changes in var gene expression, confirming that changes in SAM availability modulate var gene switching. These observations directly challenge the paradigm that antigenic variation in P. falciparum follows an intrinsic, programed switching rate, which operates independently of any external stimuli.

A nuclear redox sensor modulates gene activation and var switching in Plasmodium falciparum.

The virulence of Plasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites "choose" to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression between var genes, each encoding a different PfEMP1 variant. While the mechanisms that regulate mutually exclusive expression of var genes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the active var gene were implicated in the "choice" of the single active var gene. Here, we show that this lncRNA colocalizes with the site of var mRNA transcription and is anchored to the var locus via DNA:RNA interactions. We define the var lncRNA interactome and identify a redox sensor, P. falciparum thioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with the var antisense lncRNA. We show that PfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, when var transcription occurs. In addition, PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation of var2csa, similar to overexpression of PfSAMS. Furthermore, we show that PfTPx-1 knockdown alters the var switch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclear PfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.

Antibody to Plasmodium falciparum Variant Surface Antigens, var Gene Transcription, and ABO Blood Group in Children With Severe or Uncomplicated Malaria.

BACKGROUND: Antibodies to variant surface antigens (VSAs) such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) may vary with malaria severity. The influence of ABO blood group on antibody development is not understood. METHODS: Immunoglobulin G antibodies to VSAs in Papua New Guinean children with severe (n = 41) or uncomplicated (n = 30) malaria were measured by flow cytometry using homologous P falciparum isolates. Isolates were incubated with ABO-matched homologous and heterologous acute and convalescent plasma. RNA was used to assess var gene transcription. RESULTS: Antibodies to homologous, but not heterologous, isolates were boosted in convalescence. The relationship between antibody and severity varied by blood group. Antibodies to VSAs were similar in severe and uncomplicated malaria at presentation, higher in severe than uncomplicated malaria in convalescence, and higher in children with blood group O than other children. Six var gene transcripts best distinguished severe from uncomplicated malaria, including UpsA and 2 CIDRα1 domains. CONCLUSIONS: ABO blood group may influence antibody acquisition to VSAs and susceptibility to severe malaria. Children in Papua New Guinea showed little evidence of acquisition of cross-reactive antibodies following malaria. Var gene transcripts in Papua New Guinean children with severe malaria were similar to those reported from Africa.

The Rapid and Spontaneous Postpartum Clearance of Plasmodium falciparum Is Related to Expulsion of the Placenta.

Parasitemia among pregnant women with protective immunity to Plasmodium falciparum malaria is often dominated by VAR2CSA-positive infected erythrocytes (IEs). VAR2CSA mediates sequestration of IEs in the placenta. We hypothesized that the previously observed spontaneous postpartum clearance of parasitemia in such women is related to the expulsion of the placenta, which removes the sequestration focus of VAR2CSA-positive IEs. We assessed parasitemias and gene transcription before and shortly after delivery in 17 Ghanaian women. The precipitous decline in parasitemia postpartum was accompanied by selective reduction in transcription of the gene encoding VAR2CSA. Our findings provide a mechanistic explanation for the earlier observation.

Transcriptome Analysis of Plasmodium falciparum Isolates From Benin Reveals Specific Gene Expression Associated With Cerebral Malaria.

Cerebral malaria (CM) is the severest form of Plasmodium falciparum infection. Children under 5 years old are those most vulnerable to CM, and they consequently have the highest risk of malaria-related death. Parasite-associated factors leading to CM are not yet fully elucidated. We therefore sought to characterize the gene expression profile associated with CM, using RNA sequencing data from 15 CM and 15 uncomplicated malaria isolates from Benin. Cerebral malaria parasites displayed reduced circulation times, possibly related to higher cytoadherence capacity. Consistent with the latter, we detected increased var genes abundance in CM isolates. Differential expression analyses showed that distinct transcriptome profiles are signatures of malaria severity. Genes involved in adhesion, excluding variant surface antigens, were dysregulated, supporting the idea of increased cytoadhesion capacity of CM parasites. Finally, we found dysregulated expression of genes in the entry into host pathway that may reflect greater erythrocyte invasion capacity of CM parasites.

Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite.

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

Conserved associations between G-quadruplex-forming DNA motifs and virulence gene families in malaria parasites.

BACKGROUND: The Plasmodium genus of malaria parasites encodes several families of antigen-encoding genes. These genes tend to be hyper-variable, highly recombinogenic and variantly expressed. The best-characterized family is the var genes, exclusively found in the Laveranian subgenus of malaria parasites infecting humans and great apes. Var genes encode major virulence factors involved in immune evasion and the maintenance of chronic infections. In the human parasite P. falciparum, var gene recombination and diversification appear to be promoted by G-quadruplex (G4) DNA motifs, which are strongly associated with var genes in P. falciparum. Here, we investigated how this association might have evolved across Plasmodium species - both Laverania and also more distantly related species which lack vars but encode other, more ancient variant gene families. RESULTS: The association between var genes and G4-forming motifs was conserved across Laverania, spanning ~ 1 million years of evolutionary time, with suggestive evidence for evolution of the association occurring within this subgenus. In rodent malaria species, G4-forming motifs were somewhat associated with pir genes, but this was not conserved in the Laverania, nor did we find a strong association of these motifs with any gene family in a second outgroup of avian malaria parasites. Secondly, we compared two different G4 prediction algorithms in their performance on extremely A/T-rich Plasmodium genomes, and also compared these predictions with experimental data from G4-seq, a DNA sequencing method for identifying G4-forming motifs. We found a surprising lack of concordance between the two algorithms and also between the algorithms and G4-seq data. CONCLUSIONS: G4-forming motifs are uniquely strongly associated with Plasmodium var genes, suggesting a particular role for G4s in recombination and diversification of these genes. Secondly, in the A/T-rich genomes of Plasmodium species, the choice of prediction algorithm may be particularly influential when studying G4s in these important protozoan pathogens.

Identification of a conserved var gene in different Plasmodium falciparum strains.

BACKGROUND: The multicopy var gene family of Plasmodium falciparum is of crucial importance for pathogenesis and antigenic variation. So far only var2csa, the var gene responsible for placental malaria, was found to be highly conserved among all P. falciparum strains. Here, a new conserved 3D7 var gene (PF3D7_0617400) is identified in several field isolates. METHODS: DNA sequencing, transcriptional analysis, Cluster of Differentiation (CD) 36-receptor binding, indirect immunofluorescence with PF3D7_0617400-antibodies and quantification of surface reactivity against semi-immune sera were used to characterize an NF54 clone and a Gabonese field isolate clone (MOA C3) transcribing the gene. A population of 714 whole genome sequenced parasites was analysed to characterize the conservation of the locus in African and Asian isolates. The genetic diversity of two var2csa fragments was compared with the genetic diversity of 57 microsatellites fragments in field isolates. RESULTS: PFGA01_060022400 was identified in a Gabonese parasite isolate (MOA) from a chronic infection and found to be 99% identical with PF3D7_0617400 of the 3D7 genome strain. Transcriptional analysis and immunofluorescence showed expression of the gene in an NF54 and a MOA clone but CD36 binding assays and surface reactivity to semi-immune sera differed markedly in the two clones. Long-read Pacific bioscience whole genome sequencing showed that PFGA01_060022400 is located in the internal cluster of chromosome 6. The full length PFGA01_060022400 was detected in 36 of 714 P. falciparum isolates and 500 bp fragments were identified in more than 100 isolates. var2csa was in parts highly conserved (He = 0) but in other parts as variable (He = 0.86) as the 57 microsatellites markers (He = 0.8). CONCLUSIONS: Individual var gene sequences exhibit conservation in the global parasite population suggesting that purifying selection may limit overall genetic diversity of some var genes. Notably, field and laboratory isolates expressing the same var gene exhibit markedly different phenotypes.

Evidence of strain structure in Plasmodium falciparum var gene repertoires in children from Gabon, West Africa.

Existing theory on competition for hosts between pathogen strains has proposed that immune selection can lead to the maintenance of strain structure consisting of discrete, weakly overlapping antigenic repertoires. This prediction of strain theory has conceptual overlap with fundamental ideas in ecology on niche partitioning and limiting similarity between coexisting species in an ecosystem, which oppose the hypothesis of neutral coexistence. For Plasmodium falciparum, strain theory has been specifically proposed in relation to the major surface antigen of the blood stage, known as PfEMP1 and encoded by the multicopy multigene family known as the var genes. Deep sampling of the DBLα domain of var genes in the local population of Bakoumba, West Africa, was completed to define whether patterns of repertoire overlap support a role of immune selection under the opposing force of high outcrossing, a characteristic of areas of intense malaria transmission. Using a 454 high-throughput sequencing protocol, we report extremely high diversity of the DBLα domain and a large parasite population with DBLα repertoires structured into nonrandom patterns of overlap. Such population structure, significant for the high diversity of var genes that compose it at a local level, supports the existence of "strains" characterized by distinct var gene repertoires. Nonneutral, frequency-dependent competition would be at play and could underlie these patterns. With a computational experiment that simulates an intervention similar to mass drug administration, we argue that the observed repertoire structure matters for the antigenic var diversity of the parasite population remaining after intervention.

Epigenetic Players of Chromatin Structure Regulation in Plasmodium falciparum.

The protozoan parasite Plasmodium has evolved to survive in different hosts and environments. The diverse strategies of adaptation to different niches involve differential gene expression mechanisms mediated by chromatin plasticity that are poorly characterized in Plasmodium. The parasite employs a wide variety of regulatory mechanisms to complete their life cycle and survive inside hosts. Among them, epigenetic-mediated mechanisms have been implicated for controlling chromatin organization, gene regulation, morphological differentiation, and antigenic variation. The differential gene expression in parasite is largely dependent on the nature of the chromatin structure. The histone core methylation marks and methyl mark readers contribute to chromatin dynamics. Here, we review the recent developments on various epigenetic marks and its enzymes in the Plasmodium falciparum, how these marks play a key role in the regulation of transcriptional activity of variable genes and coordinate the differential gene expression. We also discuss the possible roles of these epigenetic marks in chromatin structure regulation and plasticity at various stages of its development.

Differential expression of var subgroups and PfSir2a genes in afebrile Plasmodium falciparum malaria: a matched case-control study.

BACKGROUND: Poor knowledge on the afebrile Plasmodium falciparum biology limits elimination approaches to target asymptomatic malaria. Therefore, the association of parasite factors involved in cytoadhesion, parasite multiplication and gametocyte maturation with afebrile malaria was assessed. METHODS: Plasmodium falciparum isolates were collected from febrile (axillary temperature ≥ 37.5 °C or a reported fever in the previous 24 h) and afebrile (fever neither at the visit nor in the previous 24 h) individuals residing in Southern Mozambique. var, PfSir2a and Pfs25 transcript levels were determined by reverse transcriptase quantitative PCRs (RT-qPCRs) and compared among 61 pairs of isolates matched by parasite density, age and year of sample collection. RESULTS: The level of varC and PfSir2a transcripts was higher in P. falciparum isolates from afebrile individuals (P ≤ 0.006), while varB and DC8 genes (P ≤ 0.002) were higher in isolates from individuals with febrile infections. After adjusting the analysis by area of residence, doubling the relative transcript unit (RTU) of varC and PfSir2a was associated with a 29.7 (95% CI 4.6-192.3) and 8.5 (95% CI 1.9-32.2) fold increases, respectively, of the odds of being afebrile. In contrast, doubling the RTU of varB and DC8 was associated with a 0.8 (95% CI 0.05-0.6) and 0.2 (95% CI 0.04-0.6) fold changes, respectively, of the odds of being afebrile. No significant differences were found for Pfs25 transcript levels in P. falciparum isolates from afebrile and febrile individuals. CONCLUSIONS: var and gametocyte-specific transcript patterns in febrile and afebrile infections from southern Mozambique matched by age, parasite density and recruitment period suggest similar transmissibility but differential expression of variant antigens involved in cytoadhesion and immune-evasion.

Serologic responses to the PfEMP1 DBL-CIDR head structure may be a better indicator of malaria exposure than those to the DBL-α tag.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). METHODS: A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. RESULTS: Malian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. CONCLUSIONS: Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

Antibodies to Intercellular Adhesion Molecule 1-Binding Plasmodium falciparum Erythrocyte Membrane Protein 1-DBLß Are Biomarkers of Protective Immunity to Malaria in a Cohort of Young Children from Papua New Guinea.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration to the cerebral microvasculature via binding of DBLß domains to intercellular adhesion molecule 1 (ICAM1) and is associated with severe cerebral malaria. In a cohort of 187 young children from Papua New Guinea (PNG), we examined baseline levels of antibody to the ICAM1-binding PfEMP1 domain, DBLß3PF11_0521, in comparison to four control antigens, including NTS-DBLα and CIDR1 domains from another group A variant and a group B/C variant. Antibody levels for the group A antigens were strongly associated with age and exposure. Antibody responses to DBLß3PF11_0521 were associated with a 37% reduced risk of high-density clinical malaria in the follow-up period (adjusted incidence risk ratio [aIRR] = 0.63 [95% confidence interval {CI}, 0.45 to 0.88; P = 0.007]) and a 25% reduction in risk of low-density clinical malaria (aIRR = 0.75 [95% CI, 0.55 to 1.01; P = 0.06]), while there was no such association for other variants. Children who experienced severe malaria also had significantly lower levels of antibody to DBLß3PF11_0521 and the other group A domains than those that experienced nonsevere malaria. Furthermore, a subset of PNG DBLß sequences had ICAM1-binding motifs, formed a distinct phylogenetic cluster, and were similar to sequences from other areas of endemicity. PfEMP1 variants associated with these DBLß domains were enriched for DC4 and DC13 head structures implicated in endothelial protein C receptor (EPCR) binding and severe malaria, suggesting conservation of dual binding specificities. These results provide further support for the development of specific classes of PfEMP1 as vaccine candidates and as biomarkers for protective immunity against clinical P. falciparum malaria.

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance.

BACKGROUND: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. RESULTS: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0-3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. CONCLUSIONS: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates.

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi - macaque model systems and summarizing exciting new progress in this area of research.

Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

Binding of Plasmodium falciparum to CD36 can be shielded by the glycocalyx.

BACKGROUND: Plasmodium falciparum-infected erythrocytes sequester in the microcirculation due to interaction between surface-expressed parasite proteins and endothelial receptors. Endothelial cells are covered in a carbohydrate-rich glycocalyx that shields against undesired leukocyte adhesion. It was investigated if the cellular glycocalyx affects the binding of P. falciparum-infected erythrocytes to CD36 in vitro. METHODS: Glycocalyx growth was followed in vitro by using azido sugars and cationized ferritin detecting O-glycoproteins and negatively charged proteoglycans, respectively. P. falciparum (clone FCR3/IT) was selected on Chinese hamster ovary (CHO) cells transfected with human CD36. Cytoadhesion to CHO CD36 at 1-4 days after seeding was quantified by using a static binding assay. RESULTS: The glycocalyx thickness of CHO cells increased during 4 days in culture as assessed by metabolic labelling of glycans with azido sugars and with electron microscopy studying the binding of cationized ferritin to cell surfaces. The functional importance of this process was addressed in binding assays by using CHO cells transfected with CD36. In parallel with the maturation of the glycocalyx, antibody-binding to CD36 was inhibited, despite stable expression of CD36. P. falciparum selected for CD36-binding recognized CD36 on CHO cells on the first day in culture, but the binding was lost after 2-4 days. CONCLUSION: The endothelial glycocalyx affects parasite cytoadhesion in vitro, an effect that has previously been ignored. The previously reported loss of glycocalyx during experimental malaria may play an important role in the pathogenesis of malaria complications by allowing the close interaction between infected erythrocytes and endothelial receptors.

Sporozoite Route of Infection Influences In Vitro var Gene Transcription of Plasmodium falciparum Parasites From Controlled Human Infections.

BACKGROUND: Antigenic variation in Plasmodium falciparum is mediated by the multicopy var gene family. Each parasite possesses about 60 var genes, and switching between active var loci results in antigenic variation. In the current study, the effect of mosquito and host passage on in vitro var gene transcription was investigated. METHODS: Thirty malaria-naive individuals were inoculated by intradermal or intravenous injection with cryopreserved, isogenic NF54 P. falciparum sporozoites (PfSPZ) generated from 1 premosquito culture. Microscopic parasitemia developed in 22 individuals, and 21 in vitro cultures were established. The var gene transcript levels were determined in early and late postpatient cultures and in the premosquito culture. RESULTS: At the early time point, all cultures preferentially transcribed 8 subtelomeric var genes. Intradermal infections had higher var gene transcript levels than intravenous infections and a significantly longer intrahost replication time (P = .03). At the late time point, 9 subtelomeric and 8 central var genes were transcribed at the same levels in almost all cultures. Premosquito and late postpatient cultures transcribed the same subtelomeric and central var genes, except for var2csa CONCLUSIONS: The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth.

Recombination events among virulence genes in malaria parasites are associated with G-quadruplex-forming DNA motifs.

BACKGROUND: Malaria parasites of the genus Plasmodium possess large hyper-variable families of antigen-encoding genes. These are often variantly-expressed and are major virulence factors for immune evasion and the maintenance of chronic infections. Recombination and diversification of these gene families occurs readily, and may be promoted by G-quadruplex (G4) DNA motifs within and close to the variant genes. G4s have been shown to cause replication fork stalling, DNA breakage and recombination in model systems, but these motifs remain largely unstudied in Plasmodium. RESULTS: We examined the nature and distribution of putative G4-forming sequences in multiple Plasmodium genomes, finding that their co-distribution with variant gene families is conserved across different Plasmodium species that have different types of variant gene families. In P. falciparum, where a large set of recombination events that occurred over time in cultured parasites has been mapped, we found a strong spatial association between these recombination events and putative G4-forming sequences. Finally, we searched Plasmodium genomes for the three classes of helicase that can unwind G4s: Plasmodium spp. have no identifiable homologue of the highly efficient G4 helicase PIF1, but they do encode two putative RecQ helicases and one homologue of the RAD3-family helicase FANCJ. CONCLUSIONS: Our analyses, conducted at the whole-genome level in multiple species of Plasmodium, support the concept that G4s are likely to be involved in recombination and diversification of antigen-encoding gene families in this important protozoan pathogen.

Differences in PfEMP1s recognized by antibodies from patients with uncomplicated or severe malaria.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants are encoded by var genes and mediate pathogenic cytoadhesion and antigenic variation in malaria. PfEMP1s can be broadly divided into three principal groups (A, B and C) and they contain conserved arrangements of functional domains called domain cassettes. Despite their tremendous diversity there is compelling evidence that a restricted subset of PfEMP1s is expressed in severe disease. In this study antibodies from patients with severe and uncomplicated malaria were compared for differences in reactivity with a range of PfEMP1s to determine whether antibodies to particular PfEMP1 domains were associated with severe or uncomplicated malaria. METHODS: Parts of expressed var genes in a severe malaria patient were identified by RNAseq and several of these partial PfEMP1 domains were expressed together with others from laboratory isolates. Antibodies from Papuan patients to these parts of multiple PfEMP1 proteins were measured. RESULTS: Patients with uncomplicated malaria were more likely to have antibodies that recognized PfEMP1 of Group C type and recognized a broader repertoire of group A and B PfEMP1s than patients with severe malaria. CONCLUSION: These data suggest that exposure to a broad range of group A and B PfEMP1s is associated with protection from severe disease in Papua, Indonesia.