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.

Highlights

  • Secreted pore-forming toxins (PFTs) are a distinct class of molecules that disrupt the barrier function through introducing large, unregulated pores in the host cell membranes [1,2,3,4,5,6]

  • The detailed compositions and physical characterization of liposomes prepared by sonication or extrusion and utilized in this work are presented in Table 1; liposome characterization by DLS provided the average diameter and polydispersity index (PDI)

  • We focused our investigations on exploring the ability of regular and longcirculating liposomes produced by either sonication or extrusion to prevent hemolysis of red blood cell (RBC) exposed to lysenin

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Summary

Introduction

Secreted pore-forming toxins (PFTs) are a distinct class of molecules that disrupt the barrier function through introducing large, unregulated pores in the host cell membranes [1,2,3,4,5,6]. Prior in vivo experiments showed that long-circulating, SM-rich liposomes had a half-maximal decline at 4 h owing to elimination by splenic macrophages, which may lead to the requirement of multiple treatments with liposomes at short time intervals [28] This may limit the applicability of liposomes to effectively remove only toxins for which SM is a preferred target. As PEG addition is intended to minimize undesired interactions, we asked whether or not such liposomes are efficient targets for toxins, actively contributing to their clearance To answer this question, we investigated the ability of PEGylated liposomes (long-circulating liposomes) to prevent RBC lysis elicited by two potent poreforming toxins, SLO [44,45] and lysenin [46,47,48]. The analysis of the inhibition plots revealed that the toxin binding to target membranes is a cooperative process influenced by the toxin’s identity and lipid composition of the liposomes

Materials
Liposome Preparation and Characterization
Red Blood Cell Preparation
Toxin Preparation
Determination of Hemolysis Rate
Method
Regular
RBCs samples
Optimization
40 H μL positive controls of 40 μL
Spectroscopic comparison of Streptolysin
Hemolysis increasing amounts of regular produced by soniFigure
Cholesterol-Free Liposomes Do Not Prevent SLO-Induced Hemolysis
Conclusions
Full Text
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