Extracellular Nucleases of Streptococcus equi subsp. zooepidemicus Degrade Neutrophil Extracellular Traps and Impair Macrophage Activity of the Host
Authors: Ma Fang , Guo Xiao , Fan Hongjie 
Published: November 2016

The pathogen
Streptococcus 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 and
S. equi
subsp.
zooepidemicus
and how these interactions explain the wide host spectrum of
S. 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 the
SESEC_RS04165
gene and the
SESEC_RS05720
gene (named
ENuc
and
5Nuc
, respectively), and three related gene deletion mutant strains, specifically, the single-mutant
ΔENuc
and
Δ5Nuc
strains and the double-mutant
ΔENuc Δ5Nuc
strain, were constructed. The
ΔENuc
and
Δ5Nuc
single-mutant strains and the
ΔENuc Δ5Nuc
double-mutant strain demonstrated lower virulence than wild-type
S. equi
subsp.
zooepidemicus
when the mouse survival rate was evaluated postinfection. Furthermore, wild-type
S. equi
subsp.
zooepidemicus
more frequently traversed the bloodstream and transferred to other organs. Wild-type
S. equi
subsp.
zooepidemicus
induced fewer NETs and was able to survive in NETs, whereas only 40% of the
ΔENuc 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-mutant
ΔENuc 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 how
S. 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 for
S. 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.