Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria.

Genome-wide association studies for severe malaria (SM) have identified 30 genetic variants mostly located in non-coding regions. Here, we aimed to identify potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium (LD) with the malaria-associated genetic variants. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing five ATP2B4 SNPs in LD with rs10900585. We found significant associations between SM and rs10900585 and our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we demonstrated that both individual SNPs and the combination of SNPs had regulatory effects. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Our data demonstrate that severe malaria-associated genetic variants alter the expression of ATP2B4 encoding a plasma membrane calcium-transporting ATPase 4 (PMCA4) expressed on red blood cells. Altering the activity of this regulatory element affects the risk of SM, likely through calcium concentration effect on parasitaemia.

CD44, a signal receptor for the inhibition of the cytoadhesion of CD36-binding Plasmodium falciparum-infected erythrocytes by CSA-binding infected erythrocytes.

The cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) in organ microvessels is a key event in the pathogenesis of cerebral malaria and pulmonary edema. Identification of the molecules involved in the interaction between IEs and endothelial cells has been a major goal of research into severe forms of malaria. In contrast, the consequences of cytoadhesion for endothelial cells have been largely ignored. By combining phenotypic selection, cytoadhesion assays and flow cytometry, we demonstrated that the cytoadhesion of CSA-binding IEs inhibited the cytoadhesion of CD36-binding IEs. We identified CD44 as a signal receptor for CSA-binding IEs cytoadhesion, and demonstrated that the signal was transduced to CD36 through a pathway involving the Src-kinase family and MEK. CD36-mediated cytoadhesion was modulated independently of changes in CD36 expression. These results provide the first evidence that some IEs can downregulate the cytoadhesion of IEs of another phenotype, by modifying endothelial cells via a signaling pathway relating CD44 to CD36. Mimicking this phenomenon may constitute an interesting therapeutic strategy for inhibiting the adhesion of CD36-binding IEs -- the most abundant phenotype among field isolates -- and promoting their degradation in the spleen.

Neural cell adhesion molecule, a new cytoadhesion receptor for Plasmodium falciparum-infected erythrocytes capable of aggregation.

The cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial cells lining the microvasculature, clogging the microvessels of various organs, is a key event in the pathogenesis of certain severe forms of malaria, such as cerebral malaria and pulmonary edema. Studies aiming to identify possible correlations between the severity of clinical cases and the presence of particular cytoadhesion phenotypes have been largely unsuccessful. One of the possible reasons for this failure is that some of the key receptors and/or mechanisms involved have yet to be identified. By combining IE selection, cell transfection, and adhesion inhibition assays, we identified a new cytoadhesion receptor, neural cell adhesion molecule (NCAM). NCAM is a member of the immunoglobulin superfamily and has nonpolysialylated and polysialylated isoforms, the latter being rare in adults. The nonpolysialylated form is present on the surfaces of endothelial cells in the microvessels of various organs in which IE sequestration occurs. We found that multiphenotypic IEs interacted with nonpolysialylated NCAM and with another, as yet unidentified receptor. These IEs also displayed cytoadhesion in flow conditions, presenting the unique ability to form adherent macroaggregates composed of hundreds of IEs. These features may act as virulence factors, increasing the capacity of IEs to clog microvessels via receptor synergy and macroaggregate formation, thereby facilitating the pathogenesis of severe forms of malaria.

Dissection of the role of PfEMP1 and ICAM-1 in the sensing of Plasmodium-falciparum-infected erythrocytes by natural killer cells.

BACKGROUND: Host innate immunity contributes to malaria clinical outcome by providing protective inflammatory cytokines such as interferon-gamma, and by shaping the adaptive immune response. Plasmodium falciparum (Pf) is the etiologic agent of the most severe forms of human malaria. Natural Killer (NK) cells are lymphocytes of the innate immune system that are the first effectors to produce interferon-gamma in response to Pf. However, the molecular bases of Pf-NK cell recognition events are unknown. Our study focuses on the role of Pf erythrocyte membrane protein 1 (PfEMP1), a major Pf virulence factor. PfEMP1 is expressed on parasitized-erythrocytes and participates to vascular obstruction through the binding to several host receptors. PfEMP1 is also a pivotal target for host antibody response to Pf infection. METHODOLOGY/PRINCIPAL FINDINGS: Using genetically-engineered parasite mutant strains, a human genetic deficiency, and blocking antibodies, we identified two receptor-ligand pairs involved in two uncoupled events occurring during the sensing of Pf infection by NK cells. First, PfEMP1 interaction with one of its host receptor, chondroitin sulfate A, mediates the cytoadhesion of Pf-infected erythrocytes to human NK cell lines, but is not required for primary NK cell activation. Second, intercellular adhesion molecule-1 (ICAM-1), another host receptor for PfEMP1, is mandatory for NK cell interferon-gamma response. In this case, ICAM-1 acts via its engagement with its host ligand, LFA-1, and not with PfEMP1, consistent with the obligatory cross-talk of NK cells with macrophages for their production of interferon-gamma. CONCLUSION/SIGNIFICANCE: PfEMP1-independent but ICAM-1/LFA-1-dependent events occurring during NK cell activation by Pf highlight the fundamental role of cellular cooperation during innate immune response to malaria.

Modeling of Plasmodium falciparum-infected erythrocyte cytoadhesion in microvascular conditions: chondroitin-4-sulfate binding, A competitive phenotype.

Although chondroitin-4-sulfate (CSA) is expressed throughout the microvasculature and CSA-binding infected erythrocytes (IE(CSA)) cytoadhere to lung and brain endothelial cells and sequester in male Saimiri sciureus, this phenotype seems to be dependent on the presence of a placenta to develop. This contradiction was investigated by modeling the interactions and cytoadhesion parameters in the microvasculature. Mixtures of IEs interacting with CSA, CD36, or intercellular adhesion molecule 1 were incubated with endothelial cells expressing the corresponding receptors, at physiological pH, under flow conditions. By use of suspensions composed of equal proportions of the phenotypes, cytoadhesion of approximately 10 times as many IE(CSA) as of any other IE tested was observed. Adherent IE(CSA) resisted microvascular wall shear stresses 3-15 times more effectively than did the others. These results, which require confirmation with field isolates, demonstrate that the CSA phenotype is competitive and are consistent with this phenotype initiating microvessel occlusion and with CSA-mediated sequestration in microvessel conditions.

Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells.

Severe malaria is characterized by the sequestration of Plasmodium falciparum-infected erythrocytes (IEs). Because platelets can affect tumor necrosis factor (TNF)-activated endothelial cells (ECs), we investigated their role in the sequestration of IEs, using IEs that were selected because they can adhere to endothelial CD36 (IE(CD36)), a P. falciparum receptor that is expressed on platelets. The results of coincubation studies indicated that platelets can induce IE(CD36) binding to CD36-deficient brain microvascular ECs. This induced cytoadhesion resisted physiological shear stress, was increased by EC stimulation with TNF, and was abolished by anti-CD36 monoclonal antibody. Immunofluorescence and scanning electron microscopy results showed that platelets serve as a bridge between IEs and the surface of ECs and may therefore provide receptors for adhesion to microvascular beds that otherwise lack adhesion receptors. This novel mechanism of cytoadhesion may reorient the sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A, a receptor for maternal malaria: monoclonal antibodies against the native parasite ligand reveal pan-reactive epitopes in placental isolates.

Plasmodium falciparum parasites express variant adhesion molecules on the surface of infected erythrocytes (IEs), which act as targets for natural protection. Recently it was shown that IE sequestration in the placenta is mediated by binding to chondroitin sulfate A via the duffy binding-like (DBL)-gamma 3 domain of P falciparum erythrocyte membrane protein 1 (PfEMP1(CSA)). Conventional immunization procedures rarely result in the successful production of monoclonal antibodies (mAbs) against such conformational vaccine candidates. Here, we show that this difficulty can be overcome by rendering Balb/c mice B cells tolerant to the surface of human erythrocytes or Chinese hamster ovary (CHO) cells before injecting P falciparum IEs or transfected CHO cells expressing the chondroitin sulfate A (CSA)-binding domain (DBL-gamma 3) of the FCR3 var(CSA) gene. We fused spleen cells with P3U1 cells and obtained between 20% and 60% mAbs that specifically label the surface of mature infected erythrocytes of the CSA phenotype (mIE(CSA)) but not of other adhesive phenotypes. Surprisingly, 70.8% of the 43 mAbs analyzed in this work were IgM. All mAbs immunoprecipitated PfEMP1(CSA) from extracts of (125)I surface-labeled IE(CSA). Several mAbs bound efficiently to the surface of CSA-binding parasites from different geographic areas and to placental isolates from West Africa. The cross-reactive mAbs are directed against the DBL-gamma 3(CSA), demonstrating that this domain, which mediates CSA binding, is able to induce a pan-reactive immune response. This work is an important step toward the development of a DBL-gamma 3-based vaccine that could protect pregnant women from pathogenesis. )