Mapping the Structural Determinants of Quorum Sensing Activity in <i>Streptococcus sinensis</i> Via Mutational Analysis of Its Competence Stimulating Peptide

<i>Streptococcus sinensis</i> is an oral commensal bacterium implicated in the severe cardiovascular disease, infective endocarditis. In Mitis group Streptococci, including <i>S. sinensis</i>, colonization, competition, and virulence are regulated by the ComABCDE quorum sensing (QS) system. This pathway depends on secretion of the competence stimulating peptide (CSP), which activates the membrane-bound histidine kinase receptor, ComD, to induce competence gene expression through the alternative sigma factor, ComX. Targeting this system offers a promising strategy for nonlethal inhibition of bacterial communication and virulence. This study investigates the structure-activity relationship (SAR) between the <i>S. sinensis</i> CSP and its ComD receptor. Alanine- and d-amino acid-substituted CSP libraries were synthesized and screened using a <i>PcomX</i>-luciferase reporter strain to assess receptor activation. Key residues required for ComD binding and activation were identified. The <i>N</i>-terminal aspartic acid was essential for activation, and its substitution (CSP-D1A) yielded a potent competitive inhibitor. Circular dichroism spectroscopy revealed that CSP and most analogs adopt weak or transient α-helical structures in membrane-mimicking environments. AlphaFold 3 Multimer modeling of CSP/ComD and CSP-D1A/ComD interactions corroborated the SAR competitive inhibition findings, yet superimposition of the two predicted complexes did not indicate major differences in the binding orientation or receptor conformation, illustrating the limitations of static models when predicting structural dynamics. Overall, our findings define key molecular determinants of <i>S. sinensis</i> CSP signaling and provide a foundation for designing peptide-based QS inhibitors as novel antivirulence therapeutics.