Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal-and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.virulence factor ͉ Streptococcus pyogenes ͉ neutrophil extracellular traps M any pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. It has been hypothesized that secretion of DNase provides a growth advantage by enlarging the pool of available nucleotides by DNA hydrolysis (1). Extracellular DNase activity also has been hypothesized to play a role in the dissemination and spread of infecting bacteria by liquifying pus (2-6). In addition, a recent study implied a role for extracellular DNases in the evasion of the innate immune response by degrading neutrophil extracellular traps (NETs). NETs trap and kill bacteria extracellularly, are composed of chromatin and granule proteins, and their structure is dissipated by DNase activity (7). The extracellular bactericidal activity of neutrophils directed against Shigella flexneri and Staphylococcus aureus was reduced greatly when incubated with exogenously added DNase (7).Group A Streptococcus (GAS) is a bacterial pathogen responsible for many serious human diseases (8, 9). The wide diversity of disease manifestations caused by GAS infection is thought to be due in part to the variable number of secreted and cell-wall anchored virulence factors produced by this pathogen. These factors include capsule, M protein, streptococcal inhibitor of complement, streptococc...

Identification of the genetic events that contribute to host-pathogen interactions is important for understanding the natural history of infectious diseases and developing therapeutics. Transcriptome studies conducted on pathogens have been central to this goal in recent years. However, most of these investigations have focused on specific end points or disease phases, rather than analysis of the entire time course of infection. To gain a more complete understanding of how bacterial gene expression changes over time in a primate host, the transcriptome of group A Streptococcus (GAS) was analyzed during an 86-day infection protocol in 20 cynomolgus macaques with experimental pharyngitis. The study used 260 custom Affymetrix (Santa Clara, CA) chips, and data were confirmed by TaqMan analysis. Colonization, acute, and asymptomatic phases of disease were identified. Successful colonization and severe inflammation were significantly correlated with an early onset of superantigen gene expression. The differential expression of two-component regulators covR and spy0680 (M1 spy0874) was significantly associated with GAS colony-forming units, inflammation, and phases of disease. Prophage virulence gene expression and prophage induction occurred predominantly during high pathogen cell densities and acute inflammation. We discovered that temporal changes in the GAS transcriptome were integrally linked to the phase of clinical disease and host-defense response. Knowledge of the gene expression patterns characterizing each phase of pathogen-host interaction provides avenues for targeted investigation of proven and putative virulence factors and genes of unknown function and will assist vaccine research.