Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.

Spontaneous parametric down conversion in a doubly resonant one-dimensional photonic crystal.

We study spontaneous parametric down conversion (SPDC) in a one-dimensional photonic crystal designed to operate in a doubly resonant configuration, where the frequencies of the pump and the generated photons are both tuned to band-edge resonances. We investigate the spectral correlations of the generated photons as a function of the spectral width of the pump, and demonstrate that the SPDC generation rate can scale with the fifth power of the structure length in the limit of a quasi-continuous-wave pump. We show that such an unusual scaling can be simply connected with the scaling of second-harmonic generation in the same structure, illustrating the general link between spontaneous and stimulated parametric nonlinear processes.

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion.

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase.

The nucleus plays a central role in several key cellular processes, including chromosome organisation, DNA replication and gene transcription. Recent work suggests an association between nuclear mechanics and cell-cycle progression, but many aspects of this connection remain unexplored. Here, by monitoring nuclear shape fluctuations at different cell cycle stages, we uncover increasing inward fluctuations in late G2 and in early prophase, which are initially transient, but develop into instabilities when approaching the nuclear-envelope breakdown. We demonstrate that such deformations correlate with chromatin condensation by perturbing both the chromatin and the cytoskeletal structures. We propose that the contrasting forces between an extensile stress and centripetal pulling from chromatin condensation could mechanically link chromosome condensation with nuclear-envelope breakdown, two main nuclear processes occurring during mitosis.

The structure of a Plasmodium vivax Tryptophan Rich Antigen domain suggests a lipid binding function for a pan-Plasmodium multi-gene family.

Tryptophan Rich Antigens (TRAgs) are encoded by a multi-gene family found in all Plasmodium species, but are significantly expanded in P. vivax and closely related parasites. We show that multiple P. vivax TRAgs are expressed on the merozoite surface and that one, PVP01_0000100 binds red blood cells with a strong preference for reticulocytes. Using X-ray crystallography, we solved the structure of the PVP01_0000100 C-terminal tryptophan rich domain, which defines the TRAg family, revealing a three-helical bundle that is conserved across Plasmodium and has structural homology with lipid-binding BAR domains involved in membrane remodelling. Biochemical assays confirm that the PVP01_0000100 C-terminal domain has lipid binding activity with preference for sulfatide, a glycosphingolipid present in the outer leaflet of plasma membranes. Deletion of the putative orthologue in P. knowlesi, PKNH_1300500, impacts invasion in reticulocytes, suggesting a role during this essential process. Together, this work defines an emerging molecular function for the Plasmodium TRAg family.

Biophysical Tools and Concepts Enable Understanding of Asexual Blood Stage Malaria.

Forces and mechanical properties of cells and tissues set constraints on biological functions, and are key determinants of human physiology. Changes in cell mechanics may arise from disease, or directly contribute to pathogenesis. Malaria gives many striking examples. Plasmodium parasites, the causative agents of malaria, are single-celled organisms that cannot survive outside their hosts; thus, thost-pathogen interactions are fundamental for parasite's biological success and to the host response to infection. These interactions are often combinations of biochemical and mechanical factors, but most research focuses on the molecular side. However, Plasmodium infection of human red blood cells leads to changes in their mechanical properties, which has a crucial impact on disease pathogenesis because of the interaction of infected red blood cells with other human tissues through various adhesion mechanisms, which can be probed and modelled with biophysical techniques. Recently, natural polymorphisms affecting red blood cell biomechanics have also been shown to protect human populations, highlighting the potential of understanding biomechanical factors to inform future vaccines and drug development. Here we review biophysical techniques that have revealed new aspects of Plasmodium falciparum invasion of red blood cells and cytoadhesion of infected cells to the host vasculature. These mechanisms occur differently across Plasmodium species and are linked to malaria pathogenesis. We highlight promising techniques from the fields of bioengineering, immunomechanics, and soft matter physics that could be beneficial for studying malaria. Some approaches might also be applied to other phases of the malaria lifecycle and to apicomplexan infections with complex host-pathogen interactions.

The erythrocyte membrane properties of beta thalassaemia heterozygotes and their consequences for Plasmodium falciparum invasion.

Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria.

The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.

Endothelial glycocalyx regulates cytoadherence in Plasmodium falciparum malaria.

Malaria is associated with significant microcirculation disorders, especially when the infection reaches its severe stage. This can lead to a range of fatal conditions, from cerebral malaria to multiple organ failure, of not fully understood pathogenesis. It has recently been proposed that a breakdown of the glycocalyx, the carbohydrate-rich layer lining the vascular endothelium, plays a key role in severe malaria, but direct evidence supporting this hypothesis is still lacking. Here, the interactions between Plasmodium falciparum infected red blood cells ( PfRBCs) and endothelial glycocalyx are investigated by developing an in vitro, physiologically relevant model of human microcirculation based on microfluidics. Impairment of the glycocalyx is obtained by enzymatic removal of sialic acid residues, which, due to their terminal location and net negative charge, are implicated in the initial interactions with contacting cells. We show a more than twofold increase of PfRBC adhesion to endothelial cells upon enzymatic treatment, relative to untreated endothelial cells. As a control, no effect of enzymatic treatment on healthy red blood cell adhesion is found. The increased adhesion of PfRBCs is also associated with cell flipping and reduced velocity as compared to the untreated endothelium. Altogether, these results provide a compelling evidence of the increased cytoadherence of PfRBCs to glycocalyx-impaired vascular endothelium, thus supporting the advocated role of glycocalyx disruption in the pathogenesis of this disease.