Elucidating the Role and Structure-Activity Relationships of the Streptococcus oligofermentans Competence-Stimulating Peptide.

Streptococcus oligofermentans is an early colonizer of the oral microbiome with documented bactericidal activity against the oral pathogen Streptococcus mutans. S. oligofermentans has been observed to possess the typical comABCDE competence regulon found within most oral streptococci; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon is yet to be explored. Herein, we have both confirmed the identity of the S. oligofermentans CSP and utilized a wide range of phenotypic assays to characterize its regulatory role in competence, biofilm formation, and hydrogen peroxide formation. To determine the importance of each amino acid residue in CSP/ComD binding, we performed systematic replacement of amino acid residues within the S. oligofermentans CSP and developed a luciferase-based reporter system to assess the ability of these mutated analogues to modulate the competence regulon. Additionally, we performed CD analysis on mutated CSP analogues to determine the correlation between the peptide secondary structure and QS activation. To further explore S. oligofermentans' potential as a biotherapeutic against S. mutans infection, lead QS activators and inhibitors were used in interspecies competition assays to assess the effect of QS modulation on interactions between these two species. Lastly, we have documented a lack of S. oligofermentans-induced cytotoxicity, highlighting the potential of this native flora as a biotherapeutic with minimal health risks.