Supplementary Material for: Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil Extracellular Traps

Neutrophils are key effectors of the host innate immune response against bacterial infection. <i>Staphylococcus aureus</i> is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of <i>S. aureus</i> nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (<i>nuc</i>-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the <i>nuc</i>-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, <i>S. aureus</i> nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that <i>S. aureus</i> nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.