Catabolite control protein A controls hydrogen peroxide production and cell death in Streptococcus sanguinis

Streptococcus sanguinis is a commensal oral bacterium producing hydrogen peroxide (H₂O₂) that is dependent on pyruvate oxidase (Spx) activity. In addition to its well-known role in bacterial antagonism during interspecies competition, H₂O₂ causes cell death in about 10% of the S. sanguinis population. As a consequence of H₂O₂-induced cell death, largely intact chromosomal DNA is released into the environment. This extracellular DNA (eDNA) contributes to the self-aggregation phenotype under aerobic conditions. To further investigate the regulation of spx gene expression, we assessed the role of catabolite control protein A (CcpA) in spx expression control. We report here that CcpA represses spx expression. An isogenic ΔccpA mutant showed elevated spx expression, increased Spx abundance, and H₂O₂ production, whereas the wild type did not respond with altered spx expression in the presence of glucose and other carbohydrates. Since H₂O₂ is directly involved in the release of eDNA and bacterial cell death, the presented data suggest that CcpA is a central control element in this important developmental process in S. sanguinis.Initial development of dental biofilms is dominated by oral streptococci, which produce specific adhesins that interact with salivary proteins bathing the teeth and oral mucosa surfaces (29). Biofilm development is a highly competitive process, and different mechanisms are used by individual bacteria to compete with other initial colonizers (17). For example, Streptococcus gordonii binding to salivary components via the surface protein Hsa has been shown to provide a competitive measure during niche competition with Streptococcus sanguinis (30). The excretion of antimicrobial components by oral streptococci as a more aggressive mode of competition has been known for several decades. Bacteriocins produced by cariogenic Streptococcus mutans are effective in inhibiting the growth of several other oral streptococci (10). Conversely, competitive hydrogen peroxide (H₂O₂) production by commensal S. sanguinis and S. gordonii during aerobic growth inhibits S. mutans (18, 20). The enzyme responsible for competitive H₂O₂ production has been identified as pyruvate oxidase (Spx, also referred to as Pox) (5, 20). Isogenic Spx⁻ mutants of S. sanguinis and S. gordonii were unable to inhibit the growth of S. mutans in an in vitro competition assay (20). A similar effective role of pyruvate oxidase dependent H₂O₂ production has been shown in the Streptococcus pneumoniae-Staphylococcus aureus interference (38). Moreover, the inverse association between S. sanguinis and more cariogenic species has been shown in clinical studies, suggesting a protective effect of S. sanguinis colonization resulting in lower caries incidence (1, 3, 6, 43). Although molecular mechanisms of this inverse relationship are not well defined, H₂O₂ production might play a role. The initial colonization process during early biofilm formation occurs when oxygen tension is high enough to allow for respiration and H₂O₂ production (25). With the consequence that H₂O₂ susceptible species might be outcompeted. This has a profound consequence on the overall composition of the biofilm because the initial colonization process influences the spatial and temporal development of the dental biofilm (15). Detailed knowledge of the regulation of pyruvate oxidase-mediated H₂O₂ production could therefore provide important insights into dental biofilm ecology and eventually lead to new ways to promote biofilm development toward a healthy composition. Initial results have shown that the pyruvate oxidases of S. sanguinis and S. gordonii are differentially regulated by glucose, despite a high homology of the promoter region. S. gordonii is not able to inhibit the growth of S. mutans in the presence of glucose, while S. sanguinis inhibiting ability is not affected (20). Furthermore, it was shown that the pyruvate oxidase dependent production of H₂O₂ is correlated with bacterial cell death and the release of extracellular DNA (eDNA). eDNA is an important component of the extracellular matrix in biofilms and in the case of S. sanguinis confers cell-cell adhesion to a certain extent, thus providing evidence that H₂O₂ production not only increases competitiveness but also promotes biofilm development (19).In this report, the regulation of pyruvate oxidase gene expression was further investigated in S. sanguinis. Carbon catabolite control protein A (CcpA) plays a role in spx expression regulation, but the regulation is not influenced by glucose. Gene expression control was also verified on the protein level. Moreover, evidence of CcpA-dependent regulation of cell death is presented in the context of increased H₂O₂ production for a ΔccpA mutant background.