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

Abstract

Streptococcus pneumoniae 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 S. pneumoniae 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 non-cognate 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 S. pneumoniae and as scaffolds for the design of QS-based anti-infective therapeutics against S. pneumoniae infections.

Development of a Dominant Negative Competence‐Stimulating Peptide (dnCSP) that Attenuates Streptococcus pneumoniae Infectivity in a Mouse Model of Acute Pneumonia

“…In a previous work, Yang et al. identified a truncated CSP2 sequence that can effectively activate the ComD2 receptor, CSP2‐desK16‐K17 (termed here CSP2(15)) . Thus, in this work, we used this truncated scaffold as the starting point for the development of pan‐group modulators.…”

“…Yang et al. have shown that replacing Asp10 in CSP2 with its d enantiomer increases the potency of the peptide by ≈20‐fold, and that a doubly mutated analogue, CSP2‐E1Ad10, is a potent ComD2 inhibitor . We therefore evaluated whether these mutations lead to the same enhancement of potency in CSP2(15).…”

“…β‐Galactosidase assay : The β‐gal assay was performed as previously described (see the Supporting Information for full details) …”

“…Previously, the Lau laboratory identified a dominant negative (dn) CSP1 analogue, CSP1‐E1A, and showed that this peptide can inhibit ComD1‐dependent virulence factor production in vitro and attenuate pherotype‐1 pneumococcus infectivity in vivo when co‐administered at the time of bacterial inoculation . More recently, the Tal‐Gan laboratory has conducted a systematic structure–activity relationship (SAR) analysis of both the CSP1 and CSP2 scaffolds and identified a potent ComD2 dnCSP: CSP2‐E1Ad10 . This analogue was also shown to weakly inhibit the ComD1 receptor, thus suggesting that CSP2 is a privileged scaffold for the design of pan‐group dnCSPs.…”

“…Our SAR analysis revealed a few positions in CSP2 that could be modified to increase the cross‐group reactivity (ComD1), while at the same time maintain the activity against the cognate receptor (ComD2). Nonetheless, because none of the new analogues was found to be a more potent ComD inhibitor than our previously reported lead ComD2 dnCSP, CSP2‐E1Ad10, we decided to evaluate the pharmacological properties and therapeutic potential of the latter. Our results show that CSP2‐E1Ad10 effectively inhibits ComD2‐mediated pneumolysin release in vitro and attenuates pherotype‐2 pneumococcus infectivity in a mouse model of acute pneumonia, even when it is administered post‐infection.…”