Red Blood Cell Susceptibility to Pneumolysin: CORRELATION WITH MEMBRANE BIOCHEMICAL AND PHYSICAL PROPERTIES

Abstract

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.

Keywords: aging; bacterial toxin; bending rigidity; diabetes; dipole potential; erythrocyte; membrane biophysics; oxidative stress; pneumolysin; surface potential.

FIGURE 1.

Variation of RBC response to PLY. A, representative phase contrast images of RBCs exposed to PLY. Scale bar, 5 μm. B, average (± S.E.) time course of hemolysis induced by PLY (118 ng/ml) in RBCs from 17 healthy individuals. C, individual time courses of hemolysis induced by PLY (118 ng/ml) in RBCs from three healthy individuals. D, dot plot of the percentage of live RBCs 30 min after exposure of RBCs from 17 healthy individuals to PLY (118 ng/ml).

FIGURE 2.

Time dependence of the normalized contour radii for two cells from the same population exposed to PLY (59 ng/ml). The radii at each time point (R_i) are normalized with respect to the values before the addition of toxin (R₀). Arrows indicate the onset of cell lysis.

FIGURE 3.

Human RBCs with prolonged resistance to eGFP-PLY show reduced toxin binding. A, representative DIC (left) and GFP fluorescence (right) images of RBCs incubated with eGFP-PLY (0.67 μg/ml). Lysed cells in the left image are characterized with faint outlines of their membranes. The cell with prolonged resistance to eGFP-PLY appears with a halo around its edge. Scale bar, 5 μm. B, correlation between GFP fluorescence intensity (hence eGFP-PLY concentration) on the surface of lysed and non-lysed cells. Each image had its background intensity subtracted, and the GFP intensities of cells (n = 38) were measured 2 min after exposure to toxin using ImageJ software (53). Significant differences are indicated: p = 0.0028 (**), unpaired, two-tailed Student t test.

FIGURE 4.

Effect of cell morphology on the susceptibility of human RBCs to PLY. A, RBCs from 17 healthy individuals were incubated with PLY (118 ng/ml), and the percentage of lysed discocytes and stomatocytes in each population was determined after exposure to PLY for 15 min. Significant differences are indicated: p = 0.0211 (*), unpaired, two-tailed Mann-Whitney t test. B, GFP fluorescence intensity of stomatocytes (n = 7) and discocytes (n = 7) after exposure to eGFP-Δ6PLY^L363A (1.12 μg/ml) for 2 min. Each image had its background intensity subtracted, and the GFP intensities of cells were measured using ImageJ software (53). Significant differences are indicated: p = 0.0012 (**), unpaired, two-tailed Mann-Whitney t test. C, merged DIC and GFP fluorescence images of echinocytes showing the distribution of eGFP-Δ6PLY^L363A on the surface of the cell. The fluorescence from eGFP-Δ6PLY^L363A is shown in red. Scale bar, 5 μm.

FIGURE 5.

Hemolysis profiles of a population of human RBCs that has undergone different biochemical treatments: ▾, control; □, H₂O₂; ▴, cumOOH; ○, DHEA; ○, calcium ionophore A23187. One representative experiment out of three is shown.

FIGURE 6.

Flow cytometry and phase contrast microscopy analysis of control and calcium ionophore A23187-treated human RBCs labeled with annexin V. A, dot plots of side-scattered (SSC-A) versus forward-scattered (FSC-A) light. One representative experiment out of three is shown. B, representative phase contrast microscopy images of control and calcium ionophore A23187-treated RBCs. Scale bar, 5 μm.

FIGURE 7.

Flow cytometry analysis of annexin V-labeled control and calcium ionophore A23187-treated RBCs before and after eGFP-Δ6PLY^L363A binding. Double fluorescence plots of red (Ex_{633 nm}/Em_{650–670 nm}, annexin V) and green fluorescence (Ex_{488 nm}/Em_{515–545 nm}, eGFP-Δ6PLY^L363A) of control, before toxin (A), calcium ionophore A23187, before toxin (B); control, after toxin (C); and calcium ionophore A23187, after toxin (D). Q1 = green; Q2 = green and red; Q3 = red; Q4 = not green and not red.

FIGURE 8.

Differences in the membrane physical properties of individual cells within a population of human RBCs are correlated with the susceptibility of cells to PLY. A, dependence of the red blood cell membrane bending elastic modulus on the time to lysis. Cells (n = 9) were exposed to 59 ng/ml PLY. One representative experiment out of three is shown. Linear regression line, R² = 0.52, p = 0.027 (*). B, dependence of the RBC membrane dipole potential on the time to lysis. Cells (n = 25) were exposed to 5.9 ng/ml PLY. One representative experiment out of three is shown. Linear regression line, R² = 0.40, p = 0.002 (**). C, dependence of the measured fluorescence intensity of FPE-labeled RBCs on the time to lysis. Cells (n = 108) were exposed to 5.9 ng/ml PLY. One representative experiment out of three is shown. Linear regression line, R² = 0.52, p = 0.029 (*).

FIGURE 9.

Correlation between levels of Hba1c and susceptibility of RBCs from individuals with diabetes to PLY. A, samples (n = 13) derived from subjects with diabetes not on metformin therapy: R² = 0.410, p = 0.018 (*). B, samples (n = 23) derived from subjects with diabetes on metformin therapy: R² = 0.054, p = 0.285 (not significant). RBCs from individuals with diabetes were exposed to PLY (118 ng/ml), and the percentage of live cells after exposure to toxin for 30 min was plotted against levels of Hba1c.

FIGURE 10.

RBCs from individuals with diabetes show increased resistance to PLY (A) and PFO (B) relative to RBCs from healthy control individuals. Average (± S.E.) time course of hemolysis induced by PLY (118 ng/ml) or PFO (72 ng/ml) in RBCs from healthy individuals (n = 17) and individuals with diabetes not on metformin therapy (n = 13). Cell lysis was monitored by phase contrast microscopy for 30 min.

FIGURE 11.

Dependence of the measured projection length (L) on the applied pressure, ΔP (L and ΔP are scaled according to Equation 2), for samples derived from subjects with diabetes (●) and from healthy controls (♦). Each point represents an average measurement for each group (n = 12 in the diabetic group and n = 14 in the healthy control group). The modulus of shear elasticity was evaluated from the gradient of the linear fits.

FIGURE 12.

Isosurface of PLY monomer. A, ribbon representation of the PLY peptide backbone with the four domains numbered. The highly conserved tryptophan-rich loop and the cholesterol binding region at the base of domain 4 are shown in stick and ball representations, respectively. The two putative sugar-binding sites, site 1 near the conserved tryptophan-rich loop (purple) and site 2 at the domain 3-domain 4 interface (gray), are shown in stick representation. B, corresponding PLY isosurface was drawn at −1 (red) and +1 (blue) contour levels as calculated and drawn using the APBS (125, 126) add-on to PyMOL (127). The field projecting from the PLY monomer is mostly, but not completely, negative.