Structure–Activity Analysis of the Competence Stimulating Peptide-1 in <i>Streptococcus mitis</i>

Antibiotic-resistant bacteria are one of the greatest challenges in modern medicine, as multidrug-resistant strains continue to outpace traditional antibiotic development. Unlike conventional antibiotics that promote resistance through selective pressure, quorum sensing-targeted therapies disrupt bacterial communication systems that regulate, among other traits, virulence, biofilm formation, and resistance acquirement through competence without directly killing the bacteria. In this study we examined quorum sensing modulation in Streptococcus mitis, a commensal oral bacterium that has recently been identified as an opportunistic pathogen capable of causing serious infections in immunocompromised individuals. The competence regulon quorum sensing system in S. mitis is controlled by the competence-stimulating peptide (CSP), which activates the histidine kinase receptor ComD to drive quorum sensing-regulated processes. S. mitis strains can be divided into different specificity groups, or pherotypes, based on the CSP they produce and cognate ComD receptor. In this work we set out to define the molecular interactions that drive CSP1/ComD1 binding and lead to ComD1 activation by conducting a systematic structure–activity analysis of the S. mitis -CSP1 sequence. To this end, we synthesized and screened two S. mitis -CSP1 analog libraries: the first, an alanine scan to identify key side-chain residues responsible for activity, and the second, a d -amino acid scan to evaluate the effect of side chain spatial orientation. Then, following the construction of a luminescence-based S. mitis NCTC 8033 quorum sensing reporter strain, we conducted reporter gene bioassays to gain insights into the structure–activity relationship between S. mitis -CSP1 and its cognate ComD1 receptor. Interestingly, our results revealed that the glutamic acid-1-to-alanine analog ( S. mitis -CSP1-E1A) had increased potency but lower efficacy, indicating partial agonist effects in S. mitis NCTC 8033, whereas the same glutamic acid-1-to-alanine substitution in CSPs from other Streptococcus species, as well as in a different S. mitis pherotype producing and responding to S. mitis -CSP2, has been reported to produce inhibitory analogs. These results suggest that the ComD1 receptor in S. mitis NCTC 8033 interacts with S. mitis -CSP1 differently than its homologues in other Streptococcus species, highlight the importance of species-specific studies, and caution against overgeneralizing bacterial behavior within a given genus. Overall, our work offers mechanistic insight into S. mitis quorum sensing signaling and lays a foundation for the rational development of CSP-based tools to study and potentially control quorum sensing-regulated processes in streptococci.