Multiple Parameters Beyond Lipid Binding Affinity Drive Cytotoxicity of Cholesterol-Dependent Cytolysins.

Sucharit Ray,Peter Keyel,Roshan Thapa

doi:10.3390/toxins11010001

Abstract

The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.

Highlights

The largest superfamily of virulence factors, utilized by many pathogenic bacteria, is the pore-forming toxins (PFTs)
To test if the differences in membrane binding between Perfringolysin O (PFO) and Streptolysin O (SLO) change the rate at which these PFTs kill nucleated cells, we challenged cells with equivalent hemolytic doses of PFO or SLO for 5 to 30 min and measured cytotoxicity by flow cytometry using propidium iodide (PI) uptake (Figure 1 and Supplemental Figure S1)
It was further important to normalize to hemolytic dose because it allowed us to compare changes in membrane repair between different toxins and their lytic mutants, without interference from overall changes in hemolytic activity due to structural changes

Summary

Introduction

The largest superfamily of virulence factors, utilized by many pathogenic bacteria, is the pore-forming toxins (PFTs). PFTs are generally classified by the secondary structure forming the pore, pore size, cellular target, and mechanism of binding. Pore insertion can lead to many deleterious effects for the target cell, including cell death, enhanced infection, depolarization, and impairment of immune activation [2,3,4,5,6]. These effects can be lethal to the host, as is the case for necrotizing soft tissue infections (NSTIs).

Methods

Results

Conclusion