Manoj T. Duraisingh

Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum undergoes antigenic variation to evade host immune responses through switching expression of variant surface proteins encoded by the var gene family. We demonstrate that both a subtelomeric transgene and var genes are subject to reversible gene silencing. Var gene silencing involves the SIR complex as gene disruption of PfSIR2 results in activation of this gene family. We also demonstrate that perinuclear gene activation involves chromatin alterations and repositioning into a location that may be permissive for transcription. Together, this implies that locus repositioning and heterochromatic silencing play important roles in the epigenetic regulation of virulence genes in P. falciparum.

Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum

Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor–ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor–ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Oka− erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor–ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCΔex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCΔex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.

A genome-wide map of diversity in Plasmodium falciparum

Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (π = 1.16 × 10−3) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.

The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin

Although chloroquine-resistance (CQR) in Plasmodium falciparum is increasing and resistance to other blood schizonticidal anti-malarials has been reported, the molecular basis remains unclear. In this study fresh field isolates were obtained from The Gambia, an area of emerging CQR and tested for sensitivity to the anti-malarial drugs mefloquine, halofantrine, artemisinin, dihydroartemisinin, chloroquine and quinine. Sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the cg2 gene were assessed using PCR-based systems. A strong association was observed between the presence of the tyr-86 allele of pfmdr1 and increased sensitivity to mefloquine and halofantrine, as well as the structurally unrelated drugs artemisinin and dihydroartemisinin. A weaker association was found between the presence of tyr-86 and increased resistance to chloroquine and quinine. The cg2 Dd2-like omega repeat size polymorphism was associated with increased resistance to chloroquine and increased sensitivity to mefloquine and halofantrine. An intragenic association was also found between a polymorphism in the polyasparagine linker region of pfmdr1 and the tyr-86 allele, which may be due to genetic hitchhiking, indicative of recent selection by chloroquine. Our data support a hypothesis where the pfmdr1 gene confers a true multidrug resistance phenotype which is lost by mutation.

Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes

The members of the phylum Apicomplexa parasitize a wide range of eukaryotic host cells. Plasmodium falciparum, responsible for the most virulent form of malaria, invades human erythrocytes using several specific and high affinity ligand–receptor interactions that define invasion pathways. We find that members of the P.falciparum reticulocyte‐binding homolog protein family, PfRh2a and PfRh2b, are expressed variantly in different lines. Targeted gene disruption shows that PfRh2b mediates a novel invasion pathway and that it functions independently of other related proteins. Phenotypic variation of the PfRh protein family allows P.falciparum to exploit different patterns of receptors on the erythrocyte surface and thereby respond to polymorphisms in erythrocyte receptors and to evade the host immune system.

Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum.

Cytoadherance of Plasmodium falciparum-infected erythrocytes in the brain, organs and peripheral microvasculature is linked to morbidity and mortality associated with severe malaria. Parasite-derived P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules displayed on the erythrocyte surface are responsible for cytoadherance and undergo antigenic variation in the course of an infection. Antigenic variation of PfEMP1 is achieved by in situ switching and mutually exclusive transcription of the var gene family, a process that is controlled by epigenetic mechanisms. Here we report characterisation of the P. falciparum silent information regulators A and B (PfSir2A and PfSir2B) and their involvement in mutual exclusion and silencing of the var gene repertoire. Analysis of P. falciparum parasites lacking either PfSir2A or PfSir2B shows that these NAD(+)-dependent histone deacetylases are required for silencing of different var gene subsets classified by their conserved promoter type. We also demonstrate that in the absence of either of these molecules mutually exclusive expression of var genes breaks down. We show that var gene silencing originates within the promoter and PfSir2 paralogues are involved in cis spreading of silenced chromatin into adjacent regions. Furthermore, parasites lacking PfSir2A but not PfSir2B have considerably longer telomeric repeats, demonstrating a role for this molecule in telomeric end protection. This work highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing of P. falciparum virulence genes and the control of pathogenicity of malaria infection.

A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes.

Its a Knockout The malaria parasite is one of the most important pathogens of humans. Increasing drug-resistance is an imminent public health disaster, and we urgently need to find new drugs. The recently acquired malarial genomes provide a plethora of targets. However, due to the genetic intractability of the parasite, it has been difficult to identify essential genes in the clinically relevant blood-stage of the parasite. Dvorin et al. (p. 910) investigated the function of a Plasmodium falciparum plant-like calcium-dependent protein kinase, PfCDPK5, which is expressed in the invasive blood-stage forms of the parasite. A system for conditional protein expression allowed the production of a functional knockout in the bloodstream stage of the parasite. PfCDPK5 was required for parasite egress from the human host erythrocyte, an essential step in the parasite life cycle. A calcium-dependent protein kinase is essential for blood-stage proliferation of the human malaria parasite. Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate–dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways

The Plasmodium falciparum erythrocyte-binding antigen 175 (EBA-175) is a ligand for merozoite invasion into human erythrocytes that binds to glycophorin A in a sialic acid-dependent manner. P. falciparum strain W2mef depends on sialic acid for invasion of erythrocytes, whereas 3D7 is sialic acid-independent. We generated parasites that lack expression or express truncated forms of EBA-175 in W2mef and 3D7. Lack of EBA-175 expression in W2mef parasites was associated with a switch to sialic acid-independent invasion. 3D7 parasites lacking expression of EBA-175 showed no alteration in their ability to utilize sialic acid-independent pathways. Strikingly, both W2mef and 3D7 parasites lacking EBA-175 expression invaded chymotrypsin-treated erythrocytes inefficiently compared with the parental lines. This loss of function suggests that the EBA-175/glycophorin A ligand–receptor interaction is the major chymotrypsin-resistant invasion pathway. Parasite lines with truncated EBA-175 had invasion phenotypes equivalent to parasites lacking expression of EBA-175. The EBA-175 ligand is functional in erythrocyte invasion by merozoites that utilize either sialic acid-dependent or -independent invasion pathways. This finding suggests a model where a minimal affinity supplied by multiple ligand–receptor interactions is required for successful invasion and has implications for EBA-175 as a malaria vaccine candidate.

Efficacy of artesunate plus pyrimethamine-sulphadoxine for uncomplicated malaria in Gambian children: a double-blind, randomised, controlled trial

BACKGROUND Resistance to cheap effective antimalarial drugs, especially to pyrimethaminesulphadoxine (Fansidar), is likely to have a striking impact on childhood mortality in sub-Sharan Africa. The use of artesunate (artesunic acid) [corrected] in combination with pyrimethamine-sulphadoxine may delay or prevent resistance. We investigated the efficacy, safety, and tolerability of this combined treatment. METHODS We did a double-blind, randomised, placebo-controlled trial in The Gambia. 600 children with acute uncomplicated Plasmodium falciparum malaria, aged 6 months to 10 years, at five health centres were randomly assigned pyrimethaminesulphadoxine (25 mg/500 mg) with placebo; pyrimethamine-sulphadoxine plus one dose of artesunate (4mg/kg bodyweight); or pyrimethamine-sulphadoxine plus one dose 4 mg/kg bodyweight artesunate daily for 3 days. Children were visited at home each day after the start of treatment until parasitaemia had cleared. FINDINGS The combined treatment was well tolerated. No adverse reactions attributable to treatment were recorded. By day 1, only 178 (47%) of 381 children treated with artesunate were still parasitaemic, compared with 157 (81%) of 195 children in the pyrimethamine-sulphadoxine alone group (relative risk 1.7 [95% CI 1.5-2.0], p<0.001). Treatment-failure rates at day 14 were 3.1% in the pyrimethamine sulphadoxine alone group, and 3.7% in the one-dose artesunate group (risk difference -0.6% [-4.2 to 3.0]) and 1.6% in the three-dose group (1.5 [1.5-4.5], p=0.048). Symptoms resolved faster in children who received artesunate, but there was no additional benefit for three doses of artesunate over one dose. Children given artesunate were less likely to be gametocytaemic after treatment. INTERPRETATION The combined treatment was safe, well tolerated, and effective. The addition of artesunate to malaria treatment regimens in Africa results in lower gametocyte rates and may lower transmission rates.