BACKGROUND Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)–propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas — one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China — with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral.

The variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on the surface of P. falciparum infected Red Blood Cells (iRBCs) is a critical virulence factor for malaria1. Each parasite encodes 60 antigenically distinct var genes encoding PfEMP1s, but during infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune evasion mechanism to avoid the host’s antibody responses2,3. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown4–7. Here we show that knocking out the P. falciparum variant-silencing SET gene (PfSETvs), which encodes an ortholog of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual iRBCs. PfSETvs-dependent H3K36me3 is present along the entire gene body including the transcription start site (TSS) to silence var genes. With low occupancy of PfSETvs at both the TSS of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long non-coding RNA (lncRNA). These results uncover a novel role of the PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologs of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1s may also be applied to the development of a malaria vaccine.

Summary Some human Plasmodium falciparum parasites, but not others, cause malaria in Aotus monkeys. To identify the basis for this variation, we crossed two clones that differ in A. nancymaae virulence and mapped inherited traits of infectivity to erythrocyte invasion by linkage analysis. A major pathway of invasion was linked to polymorphisms in a newly-identified erythrocyte binding protein, PfRH5, found in the apical region of merozoites. Polymorphisms of PfRH5 from the A. nancymaae-virulent parent (GB4) transformed the non-virulent parent (7G8) to a virulent parasite. Conversely, replacements that removed these polymorphisms from PfRH5 converted a virulent progeny clone (LC12) to a non-virulent parasite. A proteolytic fragment of PfRH5 from the infective parasites bound to A. nancymaae erythrocytes. Our results also suggest that PfRH5 is a parasite ligand for human infection, and that amino acid substitutions can cause its binding domain to recognize different human erythrocyte surface receptors.

Plasmodium falciparum invasion of human erythrocytes involves several parasite and erythrocyte receptors that enable parasite invasion by multiple redundant pathways. A key challenge to the development of effective vaccines that block parasite infection of erythrocytes is identifying the players in these pathways and determining their function. Invasion by the parasite clone, Dd2, requires sialic acid on the erythrocyte surface; Dd2/NM is a variant selected for its ability to invade neuraminidase-treated erythrocytes that lack sialic acid. The P. falciparum protein, reticulocyte homology 4 (PfRH4), is uniquely up-regulated in Dd2/NM compared with Dd2, suggesting that it may be a parasite receptor involved in invasion. The aim of the present study was to determine the role of PfRH4 in invasion of erythrocytes and to determine whether it is a target of antibody-mediated blockade and thus a vaccine candidate. We show that both native PfRH4 and a recombinant 30-kDa protein to a conserved region of PfRH4 (rRH430) bind strongly to neuraminidase-treated erythrocytes. rRH430 blocks both the erythrocyte binding of the native PfRH4 and invasion of neuraminidase-treated erythrocytes by Dd2/NM. Taken together, these results indicate that PfRH4 is a parasite receptor involved in sialic acid-independent invasion of erythrocytes. Although antibodies to rRH430 block binding of the native protein to erythrocytes, these antibodies failed to block invasion. These findings suggest that, although PfRH4 is required for invasion of neuraminidase-treated erythrocytes by Dd2/NM, it is inaccessible for antibody-mediated inhibition of the invasion process.erythrocyte invasion ͉ red cell invasion ͉ invasion pathways ͉ erythrocyte binding ͉ sialic acids U nlike Plasmodium vivax, which depends completely on the single interaction of the parasite's Duffy binding protein with the Duffy blood group antigen on erythrocytes (1, 2), Plasmodium falciparum exploits multiple parasite receptors to invade erythrocytes. The redundancy in molecular interactions allows P. falciparum to use alternate pathways for invasion of human erythrocytes. The full repertoire of parasite receptors is not yet identified, and the role in alternate invasion pathways of those identified still remains to be fully defined (3-5).Most of the parasite receptors that are known to play a role in erythrocyte binding and invasion of Plasmodium can be classified into two families. First, the Duffy binding-like (DBL) family that includes the P. vivax/Plasmodium knowlesi Duffy binding proteins and the P. falciparum erythrocyte binding-like proteins (EBA-175, BAEBL, JESEBL, EBL-1, and PEBL). Second, the reticulocyte binding-like (RBL) family that includes the Plasmodium yoelii 235-kDa rhoptry proteins, the P. vivax reticulocyte binding proteins (PvRBP-1 and -2), and the P. falciparum reticulocyte homology (PfRH) proteins (PfRH1, PfRH2a, PfRH2b, PfRH3, PfRH4, and PfRH5) (3-5).Here, we focus on one member of the PfRH family of parasite receptors, P. falciparum reticulocyte homology 4, P...

In the war against Plasmodium, humans have evolved to eliminate or modify proteins on the erythrocyte surface that serve as receptors for parasite invasion, such as the Duffy blood group, a receptor for Plasmodium vivax, and the Gerbich-negative modification of glycophorin C for Plasmodium falciparum. In turn, the parasite counters with expansion and diversification of ligand families. The high degree of polymorphism in glycophorin B found in malariaendemic regions suggests that it also may be a receptor for Plasmodium, but, to date, none has been identified. We provide evidence from erythrocyte-binding that glycophorin B is a receptor for the P.

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.

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Ma and colleagues identify CXXC5 as an epigenetic regulator required for maintaining the hypomethylation of a subset of CGIs, thereby promoting the expression of transcriptional factors such as IRF7 in pDCs to enable robust IFN response to viral infection.