Elucidating the Role of the Competence Regulon Quorum Sensing Circuitry in <i>Streptococcus cristatus</i>

<i>Streptococcus cristatus</i> belongs to the Mitis group of streptococci and is an early colonizer of the human oral cavity. This species has recently been reclassified from <i>Streptococcus oligofermentans</i>, and as such, much information regarding the competence regulon and its regulatory role in modulating downstream phenotypes remains unknown. In this work, we set out to investigate the role of the competence-stimulating peptide (CSP) in competence regulon activation and modulation, as well as define the resultant transcriptomic and phenotypic effects of CSP exposure. To this end, following confirmation of the CSP identity, structure activity relationship (SAR) analyses were conducted and revealed residues integral for CSP::ComD binding and activation, as well as provided insights about the CSP secondary structure. The ability of synthesized CSP analogs to modulate the competence regulon was quantified with the aid of a newly developed luciferase-based reporter strain, after which the biological activity was correlated with peptide secondary structure derived through CD analysis. Furthermore, RNA-seq was utilized to gain broader insights about subsequent transcriptomic changes following CSP incubation, while phenotypic assays helped with visualizing resultant expression profiles. Lastly, to further explore the potential of <i>S. cristatus</i> as a potential biotherapeutic against the oral pathogen, <i>Streptococcus mutans</i>, interspecies competition assays were used to evaluate interactions between these two species.