Synthetic Peptides Capable of Potent Multigroup Staphylococcal Quorum Sensing Activation and Inhibition in Both Cultures and Biofilm Communities.

The pathogen Staphylococcus epidermidis uses a chemical signaling process, i.e., quorum sensing (QS), to form robust biofilms and cause human infection. Many questions remain about QS in S. epidermidis, as it uses this intercellular communication pathway to both negatively and positively regulate virulence traits. Herein, we report synthetic multigroup agonists and antagonists of the S. epidermidis accessory gene regulator (agr) QS system capable of potent superactivation and complete inhibition, respectively. These macrocyclic peptides maintain full efficacy across the three major agr specificity groups, and their activity can be "mode-switched" from agonist to antagonist via subtle residue-specific structural changes. We describe the design and synthesis of these non-native peptides and demonstrate that they can appreciably decrease biofilm formation on abiotic surfaces, underscoring the potential for agr agonism as a route to block S. epidermidis virulence. Additionally, we show that both the S. epidermidis agonists and antagonists are active in S. aureus, another common pathogen with a related agr system, yet only as antagonists. This result not only revealed one of the most potent agr inhibitors known in S. aureus but also highlighted differences in the mechanisms of agr agonism and antagonism between these related bacteria. Finally, our investigations reveal unexpected inhibitory behavior for certain S. epidermidis agr agonists at sub-activating concentrations, an observation that can be leveraged for the design of future probes with enhanced potencies. Together, these peptides provide a powerful tool set to interrogate the role of QS in S. epidermidis infections and in Staphylococcal pathogenicity in general.

Characterization of an Autoinducing Peptide Signal Reveals Highly Efficacious Synthetic Inhibitors and Activators of Quorum Sensing and Biofilm Formation in Listeria monocytogenes.

Bacteria can use chemical signals to assess their local population density in a process called quorum sensing (QS). Many of these bacteria are common pathogens, including Gram-positive bacteria that utilize agr QS systems regulated by macrocyclic autoinducing peptide (AIP) signals. Listeria monocytogenes, an important foodborne pathogen, uses an agr system to regulate a variety of virulence factors and biofilm formation, yet little is known about the specific roles of agr in Listeria infection and its persistence in various environments. Herein, we report synthetic peptide tools that will enable the study of QS in Listeria. We identified a 6-mer AIP signal in L. monocytogenes supernatants and selected it as a scaffold around which a collection of non-native AIP mimics was designed and synthesized. These peptides were evaluated in cell-based agr reporter assays to generate structure-activity relationships for AIP-based agonism and antagonism in L. monocytogenes. We discovered synthetic agonists with increased potency relative to native AIP and a synthetic antagonist capable of reducing agr activity to basal levels. Notably, the latter peptide was able to reduce biofilm formation by over 90%, a first for a synthetic QS modulator in wild-type L. monocytogenes. The lead agr agonist and antagonist in L. monocytogenes were also capable of antagonizing agr signaling in the related pathogen Staphylococcus aureus, further extending their utility and suggesting different mechanisms of agr activation in these two pathogens. This study represents an important first step in the application of chemical methods to modulate QS and concomitant virulence outcomes in L. monocytogenes.

Non-Native Peptides Capable of Pan-Activating the agr Quorum Sensing System across Multiple Specificity Groups of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of hospital-acquired infections. Traditional antibiotics have significantly reduced efficacy against this pathogen due to its ability to form biofilms on abiotic surfaces and drug resistance. The accessory gene regulator (agr) quorum sensing system is directly involved in S. epidermidis pathogenesis. Activation of agr is achieved via binding of the autoinducing peptide (AIP) signal to the extracellular sensor domain of its cognate receptor, AgrC. Divergent evolution has given rise to four agr specificity groups in S. epidermidis defined by the unique AIP sequence used by each group (AIPs-I-IV) with observed cross-group activities. As agr agonism has been shown to reduce biofilm growth in S. epidermidis, the development of pan-group activators of the agr system is of interest as a potential antivirulence strategy. To date, no synthetic compounds have been identified that are capable of appreciably activating the agr system of more than one specificity group of S. epidermidis or, to our knowledge, of any of the other Staphylococci. Here, we report the characterization of the structure-activity relationships for agr agonism by S. epidermidis AIP-II and AIP-III and the application of these new SAR data and those previously reported for AIP-I for the design and synthesis of the first multigroup agr agonists. These non-native peptides were capable of inducing the expression of critical biofilm dispersal agents (i.e., phenol-soluble modulins) in cell culture and represent new tools to study the role of quorum sensing in S. epidermidis infections.