Aerotolerance and Peroxide Resistance in Peroxidase and PerR Mutants of Streptococcus pyogenes

Adaptation to an aerobic environment provides organisms with a wider selection of ecological niches for survival. The major cost associated with this evolutionary gain is constant exposure to the deleterious effects of oxygen, particularly the partially reduced forms of oxygen that include peroxide, superoxide, and hydroxyl radicals (23). In response to this toxic exposure, aerobic organisms have evolved powerful strategies to defend themselves against the damaging effects of oxygen. The group of organisms collectively known as the lactic acid bacteria provides a useful example of adaptation to the aerobic environment. Unlike organisms that have evolved a system of oxidative phosphorylation, these bacteria have an exclusively fermentative metabolism, and they lack many of the well-characterized enzymes thought to be essential for aerobic survival (13). Nevertheless, most lactic acid bacteria are aerotolerant to some degree and thus are considered facultative anaerobes. Surprisingly, growth is unaffected or even enhanced by exposure to oxygen for some species (38). Recent investigations have suggested that interaction with oxygen plays an important role in the pathogenesis of infections caused by the lactic acid bacterium Streptococcus pyogenes. This bacterium is an important human pathogen that is responsible for various diseases, including soft tissue infections that range from minor and self-limiting (pharyngitis, impetigo) to severely destructive and life-threatening (necrotizing fasciitis). Toxigenic infections (toxic shock syndrome) and several autoimmune diseases (rheumatic fever, acute postinfectious glomerulonephritis) are also associated with infection by S. pyogenes. Adhesion to host tissues is likely to be an early event in pathogenesis for all of these diseases. Expression of a major adhesin, the fibronectin-binding protein known as protein F or Sfb, is environmentally regulated, being preferentially expressed in aerobic environments (51). Regulation occurs at the level of transcription and is apparently linked to a pathway that senses oxidative stress (22). These observations suggest that S. pyogenes encounters and reacts to an aerobic environment during infection. Consistent with an at least partially aerobic lifestyle, most isolates of S. pyogenes are highly aerotolerant, and growth yields can actually be increased under aerobic conditions when grown on certain substrates (21). To thrive in an aerobic environment, it is likely that S. pyogenes must possess mechanisms for defense against reactive oxygen species. A critical issue concerns defense against hydrogen peroxide (H2O2), a highly reactive molecule that can readily diffuse across cellular membranes and oxidatively damage a number of vital cellular components, including membrane lipids, enzymes, and DNA (39, 44). Not only may S. pyogenes encounter peroxide during the infection of tissue but, like many other lactic acid bacteria, S. pyogenes also has the capacity to produce large amounts of peroxide endogenously when it is growing under aerobic conditions (21). A large body of evidence collected from analysis of multiple bacterial species has implicated catalase, a heme-containing peroxidase, as the major factor responsible for defense against peroxide (19). The importance of catalase is further emphasized by the fact that most facultative and aerobic bacteria contain multiple catalases (14). However, as is characteristic of lactic acid bacteria, S. pyogenes does not synthesize heme and lacks catalase (13). The specific mechanisms by which S. pyogenes adapts to peroxide stress are unknown. In this study, we have examined the adaptive response of S. pyogenes to peroxide stress and the potential contribution of alternative peroxidases to this response. Using a genomics-based approach, we found that S. pyogenes contains homologues of two peroxidases that have been characterized in other species. Construction and analysis of several mutants revealed that these peroxidases contribute to defense against high-level oxidative stress. In contrast, the inactivation of these genes neither impairs growth under aerobic conditions nor hinders the ability of S. pyogenes to mount an adaptive response against peroxide. Interestingly, this adaptive response was constitutively induced in mutants lacking PerR, a negative regulator of several genes, including ahpC and catalase (katA) in Bacillus subtilis. However, because these latter two genes do not appear to be essential to the S. pyogenes adaptive response, PerR may control expression of genes responsible for a potentially novel strategy for adaptation to aerobic growth environments in S. pyogenes.