Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

Abstract

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as "knobs," introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24-48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.

Keywords: coarse-grained simulations; malaria; red blood cells; shear modulus; stiffening.

Fig. 1.

A composite CG model of the human RBC membrane. (A–C) The one-agent–thick lipid bilayer model and the spectrin network model for uninfected (A1 and A2), trophozoite-stage (B1 and B2), and schizont-stage (C1 and C2) RBCs, respectively. (D) Vertical associations between the overlying lipid bilayer and the spectrin network in normal (D1) and Pf-infected (D2) membranes. (E) The composite CG model integrating the lipid bilayer and the spectrin network. Green, normal lipid agents; yellow, lipid agents representing the knobby region; red, actin oligomers; blue, ankyrins; and gray, spectrin beads.

Fig. 2.

Effects of spectrin network remodeling on the shear responses ($\dot{\gamma }=2.97\times {10}^5{\text{s}}^{-1}$). (A–C) Sheared membranes at a shear strain $\gamma =1$ for normal (A), deficient (B), and enhanced (C) spectrin networks, respectively. (D) Shear stress–strain responses of the uninfected RBC membrane, exhibiting strain-hardening behavior. (E) Shear responses of the RBC membrane with a normal lipid bilayer and a remodeled spectrin network.

Fig. 3.

The effects of knobs on the shear responses of the RBC membranes. (A and B) Simulation snapshots of sheared RBC membranes with knobs at the trophozoite and schizont stages for a shear strain $\gamma =1$. Yellow regions represent knobs. (C) Shear stress profile in the lipid bilayer at $\gamma =1$, showing that the knobby regions undergo appreciable shear stress. (D and E) Shear stress–strain responses of the RBC membrane with relevant knob sizes and densities at the trophozoite and schizont stages.

Fig. 4.

Shear stress–strain responses of uninfected (black line) and Pf-infected RBC membranes at the trophozoite (red line) and schizont (blue line) stages.

Fig. 5.

Stiffening due to the inhomogeneous strain induced by the presence of the knobs ($\alpha =0.35$). (A) Inhomogeneous shear strain distribution in the spectrin network. The shear strain in the knob-free regions is much higher than that applied, representing a stiffening mechanism due to the strain-hardening property of the spectrin network. (B) The average shear strain in the knob-free regions is ${\gamma }_F=1.44$, whereas that in the knobby regions is ${\gamma }_K=0.74$ at an applied shear strain ${\gamma }_C=1$. The shear strain in the normal spectrin network (without knobs) is homogeneous and consistent with the applied shear strain.