Pro-inflammatory interactions of streptolysin O toxin with human neutrophils in vitro

Abstract

The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca²⁺) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca²⁺concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca²⁺-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.

Keywords:

• Streptolysin O toxin
• neutrophils
• cytosolic calcium
• elastase
• NETosis
• reactive oxygen species

Author contributions

All authors contributed significantly to the conceptualization and planning of the study. DJ, AJT, RA and GRT performed laboratory investigations; DJ, AJT, RA and GRT contributed to analysis of the data and preparation of the figures, while all authors contributed to interpretation of data and compilation of the manuscript. All authors have approved this submitted version of the manuscript.

Disclosure statement

None of the authors declares a potential conflict of interest. The authors alone are responsible for the content of this manuscript.

Informed consent statement

Written informed consent was obtained from all blood donors who partook in the study.

Institutional review board statement

As noted, permission to undertake this study and to draw blood from healthy adult human volunteers was granted by the Research Ethics Committee of the Faculty of Health Sciences, University of Pretoria (Approval number: 123/2018).

Data availability statement

Data can be made available upon reasonable request.

Additional information

Funding

The study was supported from a grant awarded to Dr D. Joseph by the School of Medicine Research Committee, Faculty of Health Sciences, University of Pretoria, South Africa.