Structure Activity Relationship Study of the XIP Quorum Sensing Pheromone in Streptococcus mutans Reveal Inhibitors of the Competence Regulon

Abstract

The dental cariogenic pathogen Streptococcus mutans coordinates competence for genetic transformation via two peptide pheromones, competence stimulating peptide (CSP) and comX-inducing peptide (XIP). CSP is sensed by the comCDE system and induces competence indirectly, whereas XIP is sensed by the comRS system and induces competence directly. In chemically defined media (CDM), after uptake by oligopeptide permease, XIP interacts with the cytosolic receptor ComR to form the XIP::ComR complex that activates the expression of comX, an alternative sigma factor that initiates the transcription of late-competence genes. In this study, we set out to determine the molecular mechanism of XIP::ComR interaction. To this end, we performed systematic replacement of the amino acid residues in the XIP pheromone and assessed the ability of the mutated analogs to modulate the competence regulon in CDM. We were able to identify structural features that are important to ComR binding and activation. Our structure–activity relationship insights led us to construct multiple XIP-based inhibitors of the comRS pathway. Furthermore, when comCDE and comRS were both stimulated with CSP and XIP, respectively, a lead XIP-based inhibitor was able to maintain the inhibitory activity. Last, phenotypic assays were used to highlight the potential of XIP-based inhibitors to attenuate pathogenicity in S. mutans and to validate the specificity of these compounds to the comRS pathway within the competence regulon. The XIP-based inhibitors developed in this study can be used as lead scaffolds for the design and development of potential therapeutics against S. mutans infections.

Ribosomally synthesized peptides, foreground players in microbial interactions: recent developments and unanswered questions

“…Overall, the C-terminus of XIP is more important for activity than the N-terminus; L2, W5 on one side, and W4, L7 on the other side are most critical for binding and receptor activation, respectively. 179…”

Modulating streptococcal phenotypes using signal peptide analogues

“…Streptococcus mutans utilizes both the ComABCDE and ComRS systems to regulate competence as well as other phenotypes linked to virulence [ 73 ]. While the ComABCDE circuitry does aid in comX regulation, direct activation of this gene relies on activation of ComRS by XIP in S. mutans [ 74 ]. When the concentration of XIP reaches a minimum threshold, XIP is reimported back into the cell by the oligopeptide permease, OppA, forms a complex with ComR, and goes on to upregulate comX transcription for the entire bacterial population.…”

“…These results indicate potential involvement with both the ComRS and ComABCDE pathways. Incubation with P32 prevented the uptake of spectinomycin resistance genes ( figure 7 ) [ 74 ]. While additional experiments are needed to further elucidate methods to effectively inhibit S. mutans QS, these studies provide promising groundwork in the search to attenuate bacterial virulence and develop effective anti-infective therapeutics using peptides [ 78 ].…”

Strategies for Streptococcus mutans biofilm dispersal through extracellular polymeric substances disruption

“…Synthetic or natural analogs have been identified to achieve QS control to interfere with the signal molecule binding to receptors. Bikash and Tal‐Gan (2020) used the chemically defined media to conduct the structure‐activity relationship studies of the CSP and XIP pheromone and construct multiple CSP/XIP‐based inhibitors. Some of these inhibitors have been demonstrated to attenuate S. mutans pathogenic phenotypes.…”