Liposomes Prevent In Vitro Hemolysis Induced by Streptolysin O and Lysenin

Abstract

The need for alternatives to antibiotics in the fight against infectious diseases has inspired scientists to focus on antivirulence factors instead of the microorganisms themselves. In this respect, prior work indicates that tiny, enclosed bilayer lipid membranes (liposomes) have the potential to compete with cellular targets for toxin binding, hence preventing their biological attack and aiding with their clearance. The effectiveness of liposomes as decoy targets depends on their availability in the host and how rapidly they are cleared from the circulation. Although liposome PEGylation may improve their circulation time, little is known about how such a modification influences their interactions with antivirulence factors. To fill this gap in knowledge, we investigated regular and long-circulating liposomes for their ability to prevent in vitro red blood cell hemolysis induced by two potent lytic toxins, lysenin and streptolysin O. Our explorations indicate that both regular and long-circulating liposomes are capable of similarly preventing lysis induced by streptolysin O. In contrast, PEGylation reduced the effectiveness against lysenin-induced hemolysis and altered binding dynamics. These results suggest that toxin removal by long-circulating liposomes is feasible, yet dependent on the particular virulence factor under scrutiny.

Keywords: hemolysis; liposomes; lysenin; pore-forming toxins; streptolysin O; virulence factors.

Figure 1

Regular and long-circulating liposomes prevent lysenin-induced hemolysis. The hemolytic activity of lysenin (left tube) is canceled by the prior addition of regular (center tube) or long-circulating (right tube) liposomes prepared by sonication.

Figure 2

Spectroscopic comparison of Streptolysin O (SLO) and lysenin hemolytic activities. The concentrations of SLO (up triangles) and lysenin (down triangles) were adjusted to yield ~85% of the hemolysis achieved by a strong hypo-osmotic shock (circles). A non-exposed sample (squares) was used as a negative control. Each spectrum represents a typical run; all of the data points are experimental values with the symbols added for easy identification.

Figure 3

Hemolysis abrogation by increasing amounts of regular liposomes produced by sonication. The non-lysed control sample (1) (red blood cells (RBCs) in PBS) showed an absence of color in the supernatant in contrast to the water-lysed RBC sample (2). Near complete hemolysis was observed for RBCs exposed to lysenin alone (3). Increasing amounts of R1 liposomes (14, 56, 112, 420 and 1680 µg) added to lysenin-exposed samples (4–8) gradually inhibited hemolysis.

Figure 4

Regular and long-circulating liposomes inhibit lysenin-induced hemolysis in a concentration-dependent manner. Regular sonicated (R1, (a)) and extruded (R2, (b)) liposomes present similar EA₅₀ and cooperativity coefficients. Long-circulating sonicated (S1 (c)) and extruded (S2, (d)) liposomes present similar EA₅₀ and cooperativity coefficients, yet larger than the values determined for regular liposomes. The experimental data (symbols) are reported as mean values (±SD, n = 3); the fit with the Hill equation is shown by the dashed line.

Figure 5

Sphingomyelin (SM) is a required component of the decoy membranes. Lysenin-induced hemolysis was not prevented even by large amounts of extruded liposomes produced without SM (RC1). The experimental data (symbols) are reported as mean values (±SD, n = 3). The interrupted line was added as a visual aid.

Figure 6

Regular and long-circulating liposomes inhibit SLO-induced hemolysis in a concentration-dependent manner. Regular sonicated (R3, (a)) and extruded (R4, (b)) liposomes present similar EA50 and cooperativity coefficients. Similar inhibitory patterns are determined for long-circulating sonicated (S3, (c)) and extruded (S3, (d)) liposomes. The experimental data are reported as mean values (±SD, n = 3); the fit with the Hill equation is shown by the dashed lines.

Figure 7

Cholesterol (Chol) is an essential component for the inhibition of SLO-induced hemolysis. A 100% relative hemolysis was recorded for RBCs exposed to SLO only (a). The addition of Chol liposomes (R3) abolished hemolysis (b). In contrast, the addition of Chol-free liposomes (RC2) presented no inhibitory effects (c). The liposomes were produced by sonication and data are represented as a mean ± SD (n = 3).