Minimal Functional Scaffolds of the Streptococcus sinensis Competence-Stimulating Peptide Define the Balance between Receptor Promiscuity and Signaling Specificity

Abstract Quorum sensing (QS) enables bacteria to coordinate collective behaviors through the detection of secreted signaling molecules and represents an attractive target for anti-virulence therapeutic development. In Streptococcus sinensis, an emerging opportunistic pathogen associated with infective endocarditis, QS is mediated by a competence-stimulating peptide (CSP) that activates the membrane-bound histidine kinase receptor ComD. Although previous studies demonstrated that the S. sinensis CSP is unusually tolerant to single-amino acid substitutions, the minimum structural requirements for productive receptor activation remained unknown. Here, we systematically define the molecular determinants of S. sinensis CSP activity through peptide truncation analysis, reverse alanine scanning, and inter-species crosstalk screening. Truncation studies identified the C-terminal Lys as dispensable for activity while highlighting the critical role of the N-terminus in receptor engagement. Reverse alanine scanning revealed multiple minimal functional scaffolds that retained native levels of QS activation despite replacement of nearly one-third of the peptide sequence with alanine, demonstrating that productive signaling requires only a limited subset of native residues. Consistent with these findings, screening of CSPs from diverse Streptococcus species revealed that only a small subset activated the S. sinensis ComD receptor, and only with substantially reduced potency relative to the native CSP, indicating that receptor permissiveness remains constrained by specific sequence requirements. Finally, incorporation of a D1A substitution predictably converted agonists, including minimal functional scaffolds, into competitive antagonists while preserving their relative potency trends. The most potent antagonist also demonstrated phenotypic suppression of natural competence in S. sinensis, confirming functional inhibition of QS-controlled behavior. Together, these findings define the minimal structural features required for S. sinensis QS activation, provide new insight into how receptor promiscuity is balanced with signaling specificity, and establish a foundation for the rational design of peptide-based QS modulators.