Identification of Ligand Specificity Determinants in AgrC, the Staphylococcus aureus Quorum-sensing Receptor

Abstract

Activation of the agr system, a major regulator of staphylococcal virulence, is initiated by the binding of a specific autoinducing peptide (AIP) to the extracellular domain of AgrC, a classical receptor histidine protein kinase. There are four known agr specificity groups in Staphylococcus aureus, and we have previously localized the determinant of AIP receptor specificity to the C-terminal half of the AgrC sensor domain. We have now identified the specific amino acid residues that determine ligand activation specificity for agr groups I and IV, the two most closely related. Comparison of the AgrC-I and AgrC-IV sequences revealed a set of five divergent residues in the region of the second extracellular loop of the receptor that could be responsible. Accordingly, we exchanged these residues between AgrC-I and AgrC-IV and tested the resulting constructs for activation by the respective AIPs, measuring activation kinetics with a transcriptional fusion of blaZ to the principal agr promoter, P3. Exchange of all five residues caused a complete switch in receptor specificity. Replacement of two of the AgrC-IV residues by the corresponding residues in AgrC-I caused the receptor to be activated by AIP-I nearly as well as the wild type AgrC-I receptor. Replacement of two different AgrC-I residues by the corresponding AgrC-IV residues broadened receptor recognition specificity to include both AIPs. Various types of intermediate activity were observed with other replacement mutations. Preliminary characterization of the AgrC-I-AIP-I interaction suggests that ligand specificity may be sterically determined.

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.…”

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

“…In previous studies we and others have identified molecular determinants of ligand-mediated activation in various S. aureus AIPs (2, 4, 6) and in AgrC-I and AgrC-IV, in which critical residues in the second extracellular loop determine group specificity (7). To begin to elucidate the mechanism by which the activation signal is transmitted within the receptor, we used random mutagenesis to isolate AgrC mutants with constitutive activity, as well as those with altered specificity for divergent AIPs.…”

“…The agr autoinducing peptide (AIP) is 7-9 residues in length and contains a 5-membered ring in which the C terminus forms a thiolactone bond with a conserved, central cysteine, a structure that is critical for activity (2)(3)(4). Binding of the AIP, via a conserved C-terminal hydrophobic patch (5) and additional specific contacts (6,7), to the hexahelical transmembrane (TM) sensor domain of its cognate receptor, AgrC, results in trans-autophosphorylation of the receptor via its cytoplasmic histidine kinase (HK) domain. This is followed by phosphotransfer to the response regulator, AgrA, which binds and activates the bidirectional agr promoters, P2 and P3.…”

Structural Basis for Ligand Recognition and Discrimination of a Quorum-quenching Antibody

“…The cognate receptor for the AIPs is the transmembrane sensor kinase AgrC. The N-terminal receptor domain of AgrC is predicted to consist of six membrane-spanning helices with three extracellular loops that constitute the AIP binding site (17)(18)(19). Upon AIP binding, the C-terminal cytoplasmic kinase domain relays the signal to AgrA and phosphorylated AgrA binds to the P2 and P3 promoters to activate AIP-controlled gene expression (20).…”