To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD+-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS.

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...