Exploration of the Staphylococcus aureus agr quorum sensing system through proximity labeling.

Abstract

Staphylococcus aureus is a common commensal pathogen that has emerged as a significant threat to global health due to its predisposition towards the development of antibiotic resistance. Pathogenesis of a S. aureus infection is controlled by the accessory gene regulator (agr) quorum sensing circuit—a cell-to-cell communication system that coordinates bacterial behavior and coordinates virulence factor expression, which causes the manifestations of disease in infected patients. Despite decades of study into both Staphylococcal virulence and the agr circuit, much remains unknown about the system, including the biochemical mechanism of regulators of agr signaling and the complete biological pathway of signaling molecule synthesis and secretion. Leveraging the precision offered by an iridium-photocatalyst enabled proximity labeling strategy, this study sought to investigate cryptic or previously unknown regulators of the agr circuit. As a proof of principle, we sought to target the AgrC sensor domain for proximity labeling. This novel targeting strategy leverages the innate binding affinity of native substrates for their specific target proteins to deliver photocatalyst specifically to biologically important binding sites, hopefully enriching proximity labeling of important interactors and regulators. Via in-vitro proximity labeling optimization, this study validates that, in the context of complex membrane environments, the designed proximity labeling probe can target AgrC and label closely interacting proteins. This technique can be used to map the interactome of other proteins involved in agr signaling, including those involved in the regulation of the AIP biosynthetic pathway. Identified interacting partners could be further explored in order to determine their biological roles in the context of agr. A deeper knowledge of these networks of regulation and signaling would greatly inform our understanding of Staphylococcal biology and the pathogenesis of S. aureus infection, hopefully leading to the development of better therapeutics and improved patient outcomes. More broadly, this work establishes a technique that could be applied to the proximity labeling of any target protein with a known peptide interacting partner that is synthetically accessible.