Phenol-Soluble Modulin α Peptide Toxins from Aggressive Staphylococcus aureus Induce Rapid Formation of Neutrophil Extracellular Traps through a Reactive Oxygen Species-Independent Pathway.

Halla Björnsdottir,Marios Stylianou,Amanda Welin,Huamei Forsman,Claes Dahlgren,Felix P Klose,Anna Karlsson,Karin Christenson,Jonas Elmwall,Agnes Dahlstrand Rudin,Johan Bylund,Constantin F Urban

doi:10.3389/fimmu.2017.00257

Abstract

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.

Highlights

Staphylococcus aureus is an important pathogen that is the leading cause of bacterial infections worldwide [1]
When neutrophils are treated with the common neutrophil extracellular traps (NETs)-inducer phorbol myristate acetate (PMA), an increase in Sytox green fluorescence can be observed after 3 h (Figure 1A), which is in accordance with the time neutrophils takes to form NETs after PMA stimulation [7, 12]
Compared to PMA, the phenol-soluble modulins (PSMs) peptides gave a more rapid response and the response was of higher magnitude (Figure 1A) with signals close to the 100% lysis control

Summary

Introduction

Staphylococcus aureus is an important pathogen that is the leading cause of bacterial infections worldwide [1]. The ROS-MPOdependent NET formation is preceded by a coordinated series of cellular events, and the mechanistic details are not completely understood, it is established that the nuclear matter is decondensed before NETs are released and that the entire process takes several hours [7, 11, 12]. This type of NET formation is often called suicidal NETosis as it leads to loss of plasma membrane integrity and neutrophil death [13]. A distinct type of NET formation, “vital NETosis,” whereby the anucleated neutrophils remain intact and functional after throwing out their DNA has been described after interactions with certain bacteria and/ or bacterial products [15,16,17,18]

Methods

Results

Conclusion