Functionalized supported membranes for quantifying adhesion of P. falciparum-infected erythrocytes.

The pathology of Plasmodium falciparum malaria is largely defined by the cytoadhesion of infected erythrocytes to the microvascular endothelial lining. The complexity of the endothelial surface and the large range of interactions available for the infected erythrocyte via parasite-encoded adhesins make analysis of critical contributions during cytoadherence challenging to define. Here, we have explored supported membranes functionalized with two important adhesion receptors, ICAM1 or CD36, as a quantitative biomimetic surface to help understand the processes involved in cytoadherence. Parasitized erythrocytes bound to the receptor-functionalized membranes with high efficiency and selectivity under both static and flow conditions, with infected wild-type erythrocytes displaying a higher binding capacity than do parasitized heterozygous sickle cells. We further show that the binding efficiency decreased with increasing intermolecular receptor distance and that the cell-surface contacts were highly dynamic and increased with rising wall shear stress as the cell underwent a shape transition. Computer simulations using a deformable cell model explained the wall-shear-stress-induced dynamic changes in cell shape and contact area via the specific physical properties of erythrocytes, the density of adhesins presenting knobs, and the lateral movement of receptors in the supported membrane.

Importance of Erythrocyte Deformability for the Alignment of Malaria Parasite upon Invasion.

Invasion of erythrocytes by merozoites is an essential step for the survival and progression of malaria parasites. To invade red blood cells (RBCs), apicomplexan parasites have to adhere with their apex to the RBC membrane. This necessary apex-membrane contact (or alignment) is not immediately established because the orientation of a free merozoite with respect to the RBC membrane is random when an adhesion contact first occurs. Therefore, it has been suggested that after the initial adhesion, merozoites facilitate their proper alignment by inducing considerable membrane deformations, frequently observed before the invasion process. This proposition is based on a positive correlation between RBC membrane deformation and successful invasion; however, the role of RBC mechanics and its deformation in the alignment process remains elusive. Using a mechanically realistic model of a deformable RBC, we investigate numerically the importance of RBC deformability for merozoite alignment. Adhesion between the parasite and RBC membrane is modeled by an attractive potential that might be inhomogeneous, mimicking possible adhesion gradients at the surface of a parasite. Our results show that RBC membrane deformations are crucial for successful merozoite alignment and require interaction strengths comparable to adhesion forces measured experimentally. Adhesion gradients along the parasite body further improve its alignment. Finally, an increased membrane rigidity is found to result in poor merozoite alignment, which can be a possible reason for a reduction in the invasion susceptibility of RBCs in several blood diseases associated with membrane stiffening.

State diagram for wall adhesion of red blood cells in shear flow: from crawling to flipping.

Red blood cells in shear flow show a variety of different shapes due to the complex interplay between hydrodynamics and membrane elasticity. Malaria-infected red blood cells become generally adhesive and less deformable. Adhesion to a substrate leads to a reduction in shape variability and to a flipping motion of the non-spherical shapes during the mid-stage of infection. Here, we present a complete state diagram for wall adhesion of red blood cells in shear flow obtained by simulations, using a particle-based mesoscale hydrodynamics approach, multiparticle collision dynamics. We find that cell flipping at a substrate is replaced by crawling beyond a critical shear rate, which increases with both membrane stiffness and viscosity contrast between the cytosol and suspending medium. This change in cell dynamics resembles the transition between tumbling and tank-treading for red blood cells in free shear flow. In the context of malaria infections, the flipping-crawling transition would strongly increase the adhesive interactions with the vascular endothelium, but might be suppressed by the combined effect of increased elasticity and viscosity contrast.

Differential time-dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum-infected hemoglobinopathic erythrocytes.

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid-osmotic model on the grounds of time-resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid-osmotic model can predict time-dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model-predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid-osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum-infected erythrocytes. The contribution of vesiculation to the malaria-protective function of hemoglobin S is discussed.

Adhesion-based sorting of blood cells: an adhesive dynamics simulation study.

Blood cells can be sorted in microfluidic devices not only based on their sizes and deformability, but also based on their adhesive properties. In particular, white blood cells have been shown to be sorted out by using adhesive micropatterns made from stripes that are tilted in regard to the direction of shear flow. Here we use adhesive dynamics simulations for round cells to quantitatively investigate this effect and to predict the optimal tilt angle. We then apply our method to predict optimal sorting conditions for malaria-infected red blood cells, which like white blood cells also adhere to and roll on adhesive substrates.

Rolling Adhesion of Schizont Stage Malaria-Infected Red Blood Cells in Shear Flow.

To avoid clearance by the spleen, red blood cells infected with the human malaria parasite Plasmodium falciparum (iRBCs) adhere to the vascular endothelium through adhesive protrusions called "knobs" that the parasite induces on the surface of the host cell. However, the detailed relation between the developing knob structure and the resulting movement in shear flow is not known. Using flow chamber experiments on endothelial monolayers and tracking of the parasite inside the infected host cell, we find that trophozoites (intermediate-stage iRBCs) tend to flip due to their biconcave shape, whereas schizonts (late-stage iRBCs) tend to roll due to their almost spherical shape. We then use adhesive dynamics simulations for spherical cells to predict the effects of knob density and receptor multiplicity per knob on rolling adhesion of schizonts. We find that rolling adhesion requires a homogeneous coverage of the cell surface by knobs and that rolling adhesion becomes more stable and slower for higher knob density. Our experimental data suggest that schizonts are at the border between transient and stable rolling adhesion. They also allow us to establish an estimate for the molecular parameters for schizont adhesion to the vascular endothelium and to predict bond dynamics in the contact region.

Effect of malaria parasite shape on its alignment at erythrocyte membrane.

During the blood stage of malaria pathogenesis, parasites invade healthy red blood cells (RBC) to multiply inside the host and evade the immune response. When attached to RBC, the parasite first has to align its apex with the membrane for a successful invasion. Since the parasite's apex sits at the pointed end of an oval (egg-like) shape with a large local curvature, apical alignment is in general an energetically unfavorable process. Previously, using coarse-grained mesoscopic simulations, we have shown that optimal alignment time is achieved due to RBC membrane deformation and the stochastic nature of bond-based interactions between the parasite and RBC membrane (Hillringhaus et al., 2020). Here, we demonstrate that the parasite's shape has a prominent effect on the alignment process. The alignment times of spherical parasites for intermediate and large bond off-rates (or weak membrane-parasite interactions) are found to be close to those of an egg-like shape. However, for small bond off-rates (or strong adhesion and large membrane deformations), the alignment time for a spherical shape increases drastically. Parasite shapes with large aspect ratios such as oblate and long prolate ellipsoids are found to exhibit very long alignment times in comparison to the egg-like shape. At a stiffened RBC, a spherical parasite aligns faster than any other investigated shape. This study shows that the original egg-like shape performs not worse for parasite alignment than other considered shapes but is more robust with respect to different adhesion interactions and RBC membrane rigidities.

Stochastic bond dynamics facilitates alignment of malaria parasite at erythrocyte membrane upon invasion.

Malaria parasites invade healthy red blood cells (RBCs) during the blood stage of the disease. Even though parasites initially adhere to RBCs with a random orientation, they need to align their apex toward the membrane in order to start the invasion process. Using hydrodynamic simulations of a RBC and parasite, where both interact through discrete stochastic bonds, we show that parasite alignment is governed by the combination of RBC membrane deformability and dynamics of adhesion bonds. The stochastic nature of bond-based interactions facilitates a diffusive-like re-orientation of the parasite at the RBC membrane, while RBC deformation aids in the establishment of apex-membrane contact through partial parasite wrapping by the membrane. This bond-based model for parasite adhesion quantitatively captures alignment times measured experimentally and demonstrates that alignment times increase drastically with increasing rigidity of the RBC membrane. Our results suggest that the alignment process is mediated simply by passive parasite adhesion.

The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes.

Sickle cell trait, a common hereditary blood disorder, protects carriers from severe disease in infections with the human malaria parasite Plasmodium falciparum. Protection is associated with a reduced capacity of parasitized erythrocytes to cytoadhere to the microvascular endothelium and cause vaso-occlusive events. However, the underpinning cellular and biomechanical processes are only partly understood and the impact on endothelial cell activation is unclear. Here, we show, by combining quantitative flow chamber experiments with multiscale computer simulations of deformable cells in hydrodynamic flow, that parasitized erythrocytes containing the sickle cell haemoglobin displayed altered adhesion dynamics, resulting in restricted contact footprints on the endothelium. Main determinants were cell shape, knob density and membrane bending. As a consequence, the extent of endothelial cell activation was decreased. Our findings provide a quantitative understanding of how the sickle cell trait affects the dynamic cytoadhesion behavior of parasitized erythrocytes and, in turn, endothelial cell activation.