The Streptococcus mutans Quorum Sensing Receptor, ComD, Does Not Contribute to the Mechanism of Action of the STAMP C16G2

Objectives: The specifically targeted antimicrobial peptide (STAMP), C16G2, was developed to target the cariogenic oral pathogen Streptococcus mutans. One of the central design concepts used to create STAMPs is to link an antimicrobial peptide to a peptide that specifically targets a bacterium. C16G2 was constructed using a modified version of the S. mutans competence stimulating pheromone (CSP) as a targeting domain. CSP regulates downstream quorum sensing responses through interaction with the membrane receptor ComD. In this work we sought to determine the role of ComD in the mechanism of action of C16G2.
Methods: CSP, C16 (the CSP derived targeting sequence) and C16G2 peptides were assayed for antimicrobial activity using broth MIC and ComD interaction using an nlmA luciferase reporter. The antimicrobial activity of C16G2 against a comD mutant and over 400 S. mutans clinical isolates with heterogeneity in their comCDE operon was determined.
Results: Both CSP and C16 interacted with ComD and activated the nlmA reporter strain, but C16G2 failed to activate the reporter strain at similar concentrations. To determine if C16G2 antimicrobial activity was responsible for the lack of luciferase activity a competitive assay with CSP and C16G2 at increasing concentrations demonstrated similar signaling compared to CSP alone at sum-MIC levels of C16G2. C16G2 was found to be active against a comD deletion mutant as well as over 400 different clinical strains with genetic heterogeneity in their comCDE operon.
Conclusions: While both CSP and the CSP derived targeting peptide, C16, activate the ComD pathway C16G2 does not interact with ComD in a meaningful way to produce the observed selectively targeted antimicrobial activity.