Molecular Insights into Quorum Sensing in the Human Pathogen Pseudomonas aeruginosa from the Structure of the Virulence Regulator LasR Bound to Its Autoinducer

Abstract

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C 12 -acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an ␣-␤-␣ fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-Å resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.Evolution has endowed bacteria with a wide variety of diffusible chemical signals that are used for communication both within and between species. The most common form of intercellular communication in Gram-negative bacteria is "quorum sensing," so named because the bacteria initiate coordinated activities only when a "quorum" population density is reached (1). This synchronization of individual behavior into cooperative group activity can have many different benefits, e.g. to produce virulence factors only when the bacterial population is sufficiently dense to survive host immune responses (2-4).The first quorum sensing system described was that of Vibrio fischeri, a symbiont of the Hawaiian bobtail squid (5, 6). The squid has a "light organ" wherein it provides nutrition for V. fischeri. Upon reaching high population density the bacteria produce bioluminescence enabling the squid to cancel its own shadow and thereby avoid predation (7). The quorum sensing mechanism triggering bioluminescence depends on the bacterial synthase LuxI, which constitutively produces a signal molecule, an acylhomoserine lactone (acyl-HSL or AHL), 2 the autoinducer. The AHL diffuses through the bacterial envelope, and, upon reaching a threshold concentration, the AHL binds and activates its receptor LuxR, a transcriptional activator of the luciferase operon (1). Many different Gram-negative bacteria have now been shown to use quorum sensing systems composed of LuxI/LuxR homologues and AHL autoinduc...

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…This has been confirmed by crystal structures demonstrating that a given AHL can occupy slightly different conformations within the ligand-binding sites of different LuxR-type proteins and that conserved binding-site residues can serve distinct functionalities for ligand binding within these different receptors (178). These differences assuredly contribute to species-specific activation of a receptor by its cognate ligand and will likely facilitate the development of small-molecule modulators designed to have more restricted or more global target specificity.…”

“…Importantly, the relative inhibition potencies of the various antagonists were found to directly correlate with the progressive repositioning of the Met89 side chain, a result that helps to explain how small changes in ligand structure can induce large changes in receptor activity (202). For other AHL receptors, including TraR and LasR, the native ligands are postulated to trigger a protein folding switch that is required for proper folding of the receptor, which enhances protein stability (175)(176)(177)(178)(179). In these cases, antagonists may function by promoting the adoption of nonnative conformations which could both prevent productive DNA-protein interactions and promote receptor degradation.…”

“…This is in agreement with studies demonstrating that even minor changes in the length or substituents of the acyl tail, which contributes to the binding affinity between the receptor and the ligand (11,166,(194)(195)(196), can have large effects on its activity (11,170,197). Alteration of the chirality of the lactone ring or substitutions of part or all of the lactone head group can also alter the activity of a given molecule for a given AHL receptor (11, 118, 165-167, 196, 197), likely because of important conserved intermolecular hydrogen bonds between the AHL lactone head group and the ligand-binding pockets of AHL receptors (175)(176)(177)(178)(179)(180). In addition to promoting ligand-receptor interaction, hydrogen bonding may also help to determine the type of activity exhibited by a given compound.…”

“…The elucidation of the crystal structures of TraR (175, 176), SdiA (177), LasR (178,179), and more recently QscR (180) has provided pivotal information regarding the key interactions between LuxR-type receptors and both native and nonnative ligands. Unfortunately, because the precise binding requirements distinguishing agonists from antagonists remain unclear and appear to differ between individual receptor proteins, the crystal structures of these proteins have not been especially helpful in the rational design of AHL analogues or non-AHL-like molecules with specific agonist or antagonist activity.…”

Cyanidin inhibits quorum signalling pathway of a food borne opportunistic pathogen

“…The crystal structure of LasR receptor protein at 1.80 Å was taken from PDB database which was already reported by Bottomley et al (2007). Auto inducing domains for the transcriptional process at the domains 20 to 60 was revealed from the database (Bauer et al 2011).…”