STUDIES OF THE BIOSYNTHESIS, SIGNALING, AND MANIPULATION OF THE STAPHYLOCOCCAL agr QUORUM SENSING CIRCUIT

Abstract

Abstract

Staphylococcus aureus is a commensal pathogen that causes serious infections,and is infamous for its rapid development of antibioitic. The Accessory gene regulating (Agr) Quorum sensing (QS) system plays a central role in its virulence regulation. S. aureus secretes an auto-inducer peptide (AIP), as a signaling molecule whose concentration indicates bacterial population density. Sensing of AIP by the AgrC-AgrA two component signaling system subsequently up/down regulates virulence genes. Manipulation of the Agr system may attenuate the detrimental effect of S. aureus infections, without imposing a strong selective pressure triggering resistance development.

In AIP maturation, the N-terminal leader peptide needs to be removed. However, the identity of the relavant component for this step remains controversial. We investigated the factors for the specific cleavage, identified the cognate protease employing a photocrosslinking strategy, and verified its peptidase activity using liposome reconstitution, with synthetic substrates identical to the native substrates.

Variation in agr divides S. aureus into four subgroups, each producing a specific AIP-AgrC pair. While the timing of QS induction is known to differ among these subgroups, the molecular basis of this phenomenon is unknown. We report the successful nanodisc reconstitution of AgrC variants, and show that the variation in agonist-induced activity of AgrC accounts for these temporal differences in QS induction. We also reveal a regulatory hotspot in AgrC that controls the basal activity of its receptor histidine kinase (RHK) domain, as well as the responsiveness of the system to ligand inputs. These studies offer insights into RHK in adaptive evolution.

S. aureus colonizes and forms biofilms on surfaces. Coating surfaces with QS-manipulating molecules provides a method to control collective behavior of S. aureus. Pro- and anti-quorum-sensing molecules were covalently attached to surfaces, where they retain their influence on bacterial behaviors. Our studies highlight the potential of this approach in regulating collective behaviors of bacteria on surfaces.

Inhibitors of Agr provide a possible treatment for S. aureus infections. We report macrocyclic peptide ligands of AgrC-I selected through the RaPID system. Selected macrocyclic peptides showed neutral antagonism of AgrC-I and AgrC-II, with high potency for Agr-I, as well as partial agonism on AgrC-III.