The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.

Colonization of the anterior nares in approximately 37% of the population is a major risk factor for severe Staphylococcus aureus infections. Here we show that wall teichoic acid (WTA), a surface-exposed staphylococcal polymer, is essential for nasal colonization and mediates interaction with human nasal epithelial cells. WTA-deficient mutants were impaired in their adherence to nasal cells, and were completely unable to colonize cotton rat nares. This study describes the first essential factor for S. aureus nasal colonization.

Group A streptococcus (GAS) is a leading cause of severe, invasive human infections, including necrotizing fasciitis and toxic shock syndrome. An important element of the mammalian innate defense system against invasive bacterial infections such as GAS is the production of antimicrobial peptides (AMPs) such as cathelicidins. In this study, we identify a specific GAS phenotype that confers resistance to host AMPs. Allelic replacement of the dltA gene encoding D-alanine-D-alanyl carrier protein ligase in an invasive serotype M1 GAS isolate led to loss of teichoic acid D-alanylation and an increase in net negative charge on the bacterial surface. Compared to the wild-type (WT) parent strain, the GAS ⌬dltA mutant exhibited increased susceptibility to AMP and lysozyme killing and to acidic pH. While phagocytic uptake of WT and ⌬dltA mutants by human neutrophils was equivalent, neutrophil-mediated killing of the ⌬dltA strain was greatly accelerated. Furthermore, we observed the ⌬dltA mutant to be diminished in its ability to adhere to and invade cultured human pharyngeal epithelial cells, a likely proximal step in the pathogenesis of invasive infection. Thus, teichoic acid D-alanylation may contribute in multiple ways to the propensity of invasive GAS to bypass mucosal defenses and produce systemic infection.

Wall teichoic acids (WTAs) are major surface components of gram-positive bacteria that have recently been shown to play a key role in nasal colonization by Staphylococcus aureus. In the present study, we assessed the impact that WTAs have on endovascular infections by using a WTA-deficient S. aureus mutant (DtagO). There were no significant differences detected between the isogenic parental strain (SA113) and the DtagO mutant in polymorphonuclear leukocyte-mediated opsonophagocytosis; killing by a prototypic platelet microbicidal protein; or binding to platelets, fibronectin, or fibrinogen. However, compared with the parental strain, the DtagO mutant adhered considerably less well to human endothelial cells, especially under flow conditions (70.3% reduction;). Beads coated with WTA bound to endothelium in a dose-dependent manner, P ! .05 suggesting that WTA contributes specifically to this interaction. These in vitro data closely paralleled those from a rabbit model of infective endocarditis in which the DtagO mutant was compared with the parental strain. Clearances of staphylococcus from the bloodstream were equivalent, but the DtagO mutant showed a significantly reduced capacity to both colonize sterile cardiac vegetations ( ) and proliferate within these P ! .05 vegetations, the kidneys, and the spleen ( ). We conclude that WTA is an important factor in the P ! .001 induction and progression of endovascular S. aureus infection, likely through a specific interaction with endothelial cells.An important feature of Staphylococcus aureus bacteremia is the high frequency with which the organism spreads from the bloodstream to other targets, such as