Clostridium perfringens Phospholipase C, an Archetypal Bacterial Virulence Factor, induces the Formation of Extracellular Traps by Human Neutrophils

Abstract

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins, released to the extracellular space to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and is involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen that produces many exotoxins associated with various animal and human diseases, including the necrotizing soft tissue infection called gas gangrene. This work demonstrates that the C. perfringens toxinotype A secretome induces NETs formation (NETosis) in human neutrophils. Antibodies against the C. perfringens phospholipase C (CpPLC) completely abrogate the NETosis-inducing activity of that secretome, and the recombinant CpPLC induces NETs formation in a dose-response manner. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that help other pathogens evade NETs. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/ extracellular signal-regulated kinase (MEK/ERK) pathways, and the production of reactive oxygen species (ROS) by the xanthine oxidase (XO) and the metabolism of arachidonic acid. CpPLC was the first bacterial toxin found to be enzymatically active and is the major virulence factor in the pathogenesis of gas gangrene. This toxin drives the formation of neutrophil/platelet aggregates within the vasculature of the infected tissues, which leads to the circulation's halt and extends the anaerobic environment for C. perfringens growth. It is suggested that this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis, and then escapes and spreads to deeper tissues. Understanding the role of NETs in the thrombotic events occurring in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.