agr receptor mutants reveal distinct modes of inhibition by staphylococcal autoinducing peptides

Abstract

Through the agr quorum-sensing system, staphylococci secrete unique autoinducing peptides (AIPs) and detect their concentration via the AgrC transmembrane receptor, coordinating local bacterial population density with global changes in gene expression. Unique AIP and AgrC variants exist within and between species, and although autologous interactions lead to agr activation, heterologous interactions usually lead to cross-inhibition, resulting in natural quorum-sensing interference. To gain insight into the mechanisms responsible for these phenomena at the level of the receptor, we used random mutagenesis to isolate variants of Staphylococcus aureus AgrC-I with constitutive activity. Constitutive mutations in the sensor domain of the receptor were localized to the last transmembrane helix, whereas those in the histidine kinase domain were mostly clustered to a region near the phosphorylation site histidine. Analysis of these mutants with a range of noncognate AIPs revealed that inhibition is manifested by inverse agonism in certain heterologous pairings and by neutral antagonism in others. In addition, we isolated and characterized an AgrC sensor domain mutant with dramatically broadened activation specificity and reduced sensitivity to inhibition, identifying a single amino acid as a critical determinant of ligand-mediated inhibition. These results suggest that certain noncognate AIPs stabilize an inhibitory receptor conformation that may be a critical feature of the ligand-receptor interaction not initially appreciated in previous analyses of agr inhibition.constitutive mutant ͉ inverse agonism ͉ staphylococci ͉ quorum sensing

Probing bacterial transmembrane histidine kinase receptor–ligand interactions with natural and synthetic molecules

“…In terms of altered, rather than broadened, specificity mutants, to achieve a change in Tar specificity, 4 to 12 simultaneous point mutations were required (40). In AgrC, either multiple mutations or exchanges of significant portions of transmembrane domains were required (39,41,42). This is not the case in CqsS, because we can change any one of five amino acids (W104, S107, F162, F166, and C170) and alter the receptor specificity.…”

“…Surprisingly, this powerful approach, to our knowledge, has not been routinely adapted for studying prokaryotic two-component signal-transduction systems. The present work, together with the elegant studies on the Agr system by Muir, Novick, and coworkers (39,41,52), suggests that rationally designed ligands can be exploited to study and ultimately to control two-component histidine kinase activity.…”

“…These and our results suggest that there are multiple routes to make a "sloppier" receptor. For example, in the case of S. aureus AgrC-1, alteration of residue Ile-171 to Lys enables the receptor to respond to its cognate peptide ligand, AIP-1, as well as to additional otherwise inactive AIP peptide variants (39). Similarly, mutation of M106, L117, or M125 allowed CqsS to recognize an increased number of molecules without affecting sensitivity to CAI-1.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Geisinger et al recently examined inhibition of constitutively active AgrC mutants by a range of AIP analogues and found that heterologous AIPs can inhibit AgrC activity by distinct mechanisms. Some heterologous AIPs were competitive inhibitors of the cognate AIP, while others were found to act as inverse agonists, inducing a nonactive AgrC conformation even in the absence of activating ligand (222). This may help to explain why residues critical for activity differ between AIP groups.…”

Quorum-sensing agr mediates bacterial oxidation response via an intramolecular disulfide redox switch in the response regulator AgrA

“…In addition, it encodes AgrD, the precursor of the quorum signal that can further be processed and exported as a thiolactone-containing oligopeptide autoinducer (autoinducing peptide, AIP) by the cotranscribed AgrB. Upon binding to the extracellular sensory domain of AgrC, AIP activates the kinase activity of AgrC, subsequently leading to phosphorylation of the response regulator AgrA (13,14). Phosphorylated AgrA regulates transcription of genes encoding metabolic factors and phenol-soluble modulin (PSM) peptides (15) and, more importantly, triggers the expression of the agr operon by binding to the promoter regions P2 (for RNAII) and P3 (for RNAIII), thereby forming an autoinduction genetic circuit to ensure a timely rearrangement of target gene expression at a certain threshold level of population density.…”