Project Summary Gram-positive bacteria process and release small peptides, or “pheromones”, that act as critical signals for the induction of adaptive traits including those involved in virulence. One class of small signaling pheromones is the cyclic auto-inducing peptide (AIP), which regulates the expression of genes that orchestrate virulence and persistence in Staphylococci, Listeria, Clostridia, and Enterococci. Defects in cyclic AIP production and signaling can compromise virulence traits of these microbes, underscoring the relevance of peptide-based signaling to health and disease. Staphylococcus aureus harbors a cyclic peptide signaling system known as the accessory gene regulatory (Agr) system. This “quorum sensing” system depends on the synthesis, processing, and export of a cyclic AIP, derived from its precursor protein, AgrD, for function. AIP signaling through Agr leads to the production of S. aureus virulence factors, whereas disruption of signaling causes significant attenuation in skin and lung infection models. Despite clear connections between Agr and S. aureus pathobiology, there exist major gaps in our knowledge of the mechanics of AIP biosynthesis. Most notably: (i) the proteins needed for peptide processing of AIP have not been elucidated; (ii) a transporter for AIP or its leader peptide has not been identified; (iii) differential processing of AIP variants has not been investigated; and (iv) conservation in cyclic peptide processing events between bacterial species is not known. In this grant, we provide data related to our discovery of a putative peptidase in S. aureus, MroQ, that we hypothesize acts directly or indirectly on Agr system components to promote the final steps in the processing and/or export of AIP. The overall goals of this grant are to interrogate the previously unknown mechanics of cyclic peptide maturation in S. aureus and provide insight into the potential conservation of function in Gram positive pathogens. Aim 1 will define how MroQ promotes AIP processing, export, or both. Aim 2 will interrogate the extent with which MroQ interacts with Agr system or other membrane components and will use biochemistry to test if MroQ directly cleaves AgrD. Aim 3 will determine the extent with which MroQ promotes activity of Agr variants both within species and among other species and identify the AgrD sequence characteristics that dictate MroQ specificity.