Structure–Activity Relationships of the Competence Stimulating Peptides (CSPs) in <i>Streptococcus pneumoniae</i> Reveal Motifs Critical for Intra-group and Cross-group ComD Receptor Activation

<i>Streptococcus pneumoniae</i> is a highly recombinogenic human pathogen that utilizes the competence stimulating peptide (CSP)-based quorum sensing (QS) circuitry to acquire antibiotic resistance genes from the environment and initiate its attack on the human host. Modulation of QS in this bacterium, either inhibition or activation, can therefore be used to attenuate <i>S. pneumoniae</i> infectivity and slow down pneumococcal resistance development. In this study, we set to determine the molecular mechanism that drives CSP:receptor binding and identify CSP-based QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in the two major CSP signals (CSP1 and CSP2) and assessed the ability of the mutated analogs to modulate QS against both cognate and noncognate ComD receptors. We then evaluated the overall 3D structures of these analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. Our CD analysis revealed a strong correlation between α-helicity and bioactivity for both specificity groups (CSP1 and CSP2). Furthermore, we identified the first pan-group QS activator and the most potent group-II QS inhibitor to date. These chemical probes can be used to study the role of QS in <i>S. pneumoniae</i> and as scaffolds for the design of QS-based anti-infective therapeutics against <i>S. pneumoniae</i> infections