Extracellular Nucleases of Streptococcus equi subsp. zooepidemicus Degrade Neutrophil Extracellular Traps and Impair Macrophage Activity of the Host

The pathogenStreptococcus equi subsp.zooepidemicus is associated with a wide range of animals, including humans, and outbreaks frequently occur in pigs, equines, and goats. Thus far, few studies have assessed interactions between the host immune system andS. equi subsp.zooepidemicus and how these interactions explain the wide host spectrum ofS. equi subsp.zooepidemicus . Neutrophils, the first line of innate immunity, possess a defense mechanism called neutrophil extracellular traps (NETs), which primarily consist of DNA and granule proteins that trap bacteria via charge interactions. Extracellular nucleases play important roles in the degradation of the DNA backbone of NETs. Here, two related extracellular nucleases, nuclease and 5′-nucleotidase (named ENuc and 5Nuc, respectively, in this study), were identified as being encoded by theSESEC_RS04165 gene and theSESEC_RS05720 gene (namedENuc and5Nuc , respectively), and three related gene deletion mutant strains, specifically, the single-mutantΔENuc andΔ5Nuc strains and the double-mutantΔENuc Δ5Nuc strain, were constructed. TheΔENuc andΔ5Nuc single-mutant strains and theΔENuc Δ5Nuc double-mutant strain demonstrated lower virulence than wild-typeS. equi subsp.zooepidemicus when the mouse survival rate was evaluated postinfection. Furthermore, wild-typeS. equi subsp.zooepidemicus more frequently traversed the bloodstream and transferred to other organs. Wild-typeS. equi subsp.zooepidemicus induced fewer NETs and was able to survive in NETs, whereas only 40% of theΔENuc Δ5Nuc double-mutant cells survived.S. equi subsp.zooepidemicus degraded the NET DNA backbone and produced deoxyadenosine, primarily through the action of ENuc and/or 5Nuc. However, the double-mutantΔENuc Δ5Nuc strain lost the ability to degrade NETs into deoxyadenosine. Deoxyadenosine decreased RAW 264.7 cell phagocytosis to 40% of that of normal macrophages.IMPORTANCE Streptococcus equi subsp.zooepidemicus causes serious bacteremia in its hosts. However, little is known about howS. equi subsp.zooepidemicus interacts with the host innate immune system, particularly innate cells found in the blood.S. equi subsp.zooepidemicus is capable of evading NET-mediated killing via the actions of its potent extracellular nucleases, ENuc and 5Nuc, which directly degrade the NET DNA backbone to deoxyadenosine. In previous studies, other pathogens have required the synergism of nuclease and 5′-nucleotidase to engage in this self-protective process; however, ENuc and 5Nuc both possess nuclease activity and 5′-nucleotidase activity, highlighting the novelty of this discovery. Furthermore, deoxyadenosine impairs phagocytosis but not the intracellular bactericidal activity of macrophages. Here we describe a novel mechanism forS. equi subsp.zooepidemicus extracellular nucleases in NET degradation, which may provide new insights into the pathogen immune evasion mechanism and the prevention and treatment of bacterial disease.