Modifications in erythrocyte membrane zeta potential by Plasmodium falciparum infection

Abstract

The zeta potential (ZP) is an electrochemical property of cell surfaces that is determined by the net electrical charge of molecules exposed at the surface of cell membranes. Membrane proteins contribute to the total net electrical charge of cell surfaces and can alter ZP through variation in their copy number and changes in their intermolecular interactions. Plasmodium falciparum extensively remodels its host red blood cell (RBC) membrane by placing 'knob'-like structures at the cell surface. Using an electrophoretic mobility assay, we found that the mean ZP of human RBCs was -15.7 mV. In RBCs infected with P. falciparum trophozoites ('iRBCs'), the mean ZP was significantly lower (-14.6 mV, p<0.001). Removal of sialic acid from the cell surface by neuraminidase treatment significantly decreased the ZP of both RBCs (-6.06 mV) and iRBCs (-4.64 mV). Parasite-induced changes in ZP varied by P. falciparum clone and the presence of knobs on the iRBC surface. Variations in ZP values were accompanied by altered binding of iRBCs to human microvascular endothelial cells (MVECs). These data suggest that parasite-derived knob proteins contribute to the ZP of iRBCs, and that electrostatic and hydrophobic interactions between iRBC and MVEC membranes are involved in cytoadherence.

Figure 1

Basic concept of zeta potential (ZP). Negatively-charged red blood cells migrate toward the positive electrode upon the application of voltage between positive and negative electrodes. ZP is defined as $\zeta =A·\frac{4\pi \eta }{\epsilon }·U$ and $U=\frac{\nu }{V/L}$, where A is a constant, ζ is the zeta potential, η is the viscosity of solution, ε is the dielectric constant, and U is the electrophoretic mobility, ν is the speed of particle, V is the applied voltage, and L is the distance of electrode (Overbeek, 1952, Weiss and Woodbridge, 1967). Dielectric constant and viscosity of the buffer is approximated as those of water.

Figure 2

Zeta potential (ZP) of red blood cell (RBC) samples. (A) ZP of P. falciparum ring-infected RBCs. (B) ZP of P. falciparum trophozoite-infected RBCs. (C) ZP of P. falciparum trophozoite-infected RBCs treated with neuraminidase. Curves represent Gaussian fitting of the data. Small differences in the peak value and distribution of ZP between RBC samples from different donors may have resulted from differences in the age-distribution of their RBCs.

Figure 3

Influence of knobs on zeta potential (ZP) and cytoadherence. (A) Electron micrographs of knobby and knobless P. falciparum trophozoite-infected red blood cells (iRBCs). Panels a–d: scanning electron microscopic images. Panels e–h: transmission electron microscopic images. Bar represents 1 μm. (B, C) ZP values of iRBCs containing Indochina and KAHRP(−) 3D7 parasites, respectively, compared to mock-cultured RBC controls. (D) Relative cytoadherence of iRBCs containing knob-forming (3D7) and knobless (Indochina, KAHRP(−) 3D7) parasite clones. An average of 1.2 3D7 iRBCs were bound per MVEC. (E) Effects of neuraminidase and trypsin treatments on cytoadherence of iRBCs containing 3D7, Indochina, or KAHRP(−) 3D7 P. falciparum clones. Cytodherence levels of untreated iRBCs were set at 100%.