Mapping of quorum sensing interaction network of commensal and pathogenic staphylococci

Staphylococci utilize secreted autoinducing peptides (AIPs) to regulate group behavior through a process called quorum sensing (QS). For pathogenic staphylococci, such as <i>Staphylococcus aureus</i>, QS regulates the expression of major virulence factors, and QS inhibition has been proposed as an alternative to antibiotics for the treatment of infections with methicillin-resistant <i>S. aureus</i> (MRSA). Here, we surveyed the interaction map between QS systems of the pathogens <i>S. aureus, Staphylococcus epidermidis,</i> and <i>Staphylococcus lugdunensis</i> and all the currently known staphylococcal AIPs, covering 21 different species. We identified six of these ribosomally synthesized and post-translationally modified peptides (RiPPs) in this study and compiled the full collection of AIPs by chemical synthesis. The resulting mapping provided 280 QS interactions that were divided into human- and animal-associated staphylococci, showing substantial differences in inhibitory potencies between the groups. AIPs of the bovine-associated species <i>Staphylococcus simulans</i> displayed potential as QS inhibitors toward the investigated strains and were therefore chosen as a starting point for a structure-activity relationship study. This study provides insights into the requirements for QS interference, yielding the most potent inhibitors reported to date for <i>S. epidermidis</i> and <i>S. lugdunensis</i>. Furthermore, we tested an <i>S. simulans</i> AIP as an anti-virulence agent in an assay to assess the risk of acquired suppression of the inhibitory effect, and we established an assay set-up to successfully monitor <i>agr</i> deactivation of virulent MRSA by the QS inhibitor. Finally, a peptide was shown to attenuate skin infection caused by MRSA in a mouse model. Our results reveal a complex network of staphylococcal interactions and provide further impetus for the investigation of QS modulation in the targeting of antibiotic-resistant pathogens.IMPORTANCEBacteria from the <i>Staphylococcus</i> genus produce macrocyclic peptides, called autoinducing peptides (AIPs), used in inter-cell communication with their kin. Differences in AIP sequence and length produced by different staphylococcal species can result in communication interference, altering the physiology of co-inhabiting staphylococci in complex microbiotas. Opportunistic pathogens like <i>Staphylococcus aureus</i> regulate the expression of toxins using this peptide-mediated communication, and its inhibition has, therefore, been proposed as a strategy to target infections caused by methicillin-resistant <i>S. aureus</i> (MRSA). The systematic mapping of AIP activities, structure-activity relationship studies, and evaluation of resistance development provided in this paper, therefore, serve as a resource for the future discovery of inhibitory peptides for the investigation of bacterial communication.