Bacterial Competition: The Impact of CcpA on Oral Streptococcal H₂O₂-Release

Objectives: Pyruvate oxidase (SpxB) dependent H₂O₂ production is under control of carbon catabolite protein A (CcpA) in Streptococcus sanguinis SK36 and Streptococcus gordonii DL1. While spxB expression and thus H₂O₂ release follow classical catabolite control pattern in S. gordonii, the expression of spxB is not altered by addition of glucose in S. sanguinis. In order to elucidate the cause for these regulatory differences and its ecological impact on early phase of multi-species-biofilm formation, we investigated CcpA binding more deeply and further tested additional streptococcal isolates.
Methods: spxB and ccpA deletion mutants, ccpA-gene and spxB-promotor exchange constructs as well as spxB_p-luciferase reporter strains were analyzed in vivo. A chromogenic assay was utilized to detect and quantify H₂O₂ production. Growth inhibition assays were performed with Streptococcus mutans. EMSAs and DNA footprint analysis were done with purified CcpA and the spxB-promotor regions.
Results: Beside a known CcpA-binding site a second one was identified and verified in S. sanguinis. Luciferase assays confirmed chromogenic quantification of H₂O₂: tight repression of spxB expression in S. gordonii after addition of glucose (2%) and independent from glucose concentration (0.2% and 2%) in S. sanguinis. Neither spxB-promotor exchange nor ccpA-gene could transfer the regulation pattern from S. sanguinis to S. gordonii or vice versa. S. mutans growth inhibition was in concordance with H₂O₂ production of all strains. Comparable regulatory patterns of H₂O₂ release were found in several new streptococcal isolates.
Conclusions: The obtained data indicate that spxB regulation is most likely influenced by additional effectors in vivo. Overall, our data show that a conservation of metabolic pathways and regulatory mechanisms does not necessarily lead to similar regulation patterns. Further investigation is needed to uncover the mechanisms in the complex ecology of oral multi-species-biofilms.