| Staphylococci and other Gram-positive bacteria secrete and sense unique peptide pheromones, partaking in a molecular collective that enables detection of local cell density and sampling of the external milieu. The versatile pathogen Staphylococcus aureus uses such information to rapidly coordinate virulence factor production with population dynamics. The agr quorum sensing locus encodes the functional elements that mediate this process, including a specific autoinducing thiolactone peptide (AIP) and its cognate transmembrane receptor AgrC, a histidine protein kinase (HPK). Unique AIP and AgrC variants exist within and between species, and while autologous peptide-receptor interactions lead to agr activation, heterologous interactions generally lead to cross-inhibition, resulting in natural interference with quorum sensing. In this work, we have analyzed various components of agr signal transduction in an effort to understand how activation and inhibition signals are generated. (i) Beginning with the AIP-AgrC interaction, we identified specific residues in the extracellular loops of the receptor that mediate either activation by cognate peptides or inhibition by noncognate ones. (ii) Exploring the processes induced upon ligand binding, we isolated constitutive AgrC variants, localizing critical residues involved in receptor activation and enabling an exploration of the conformational changes defining this process. Analysis of these mutants with a range of AIPs revealed further insights into inhibition, with the central observation that certain heterologous AIPs are inverse agonists, inducing specific receptor states associated with reversal of constitutivity. (iii) We next analyzed pairs of AgrC mutants with complementary cytoplasmic signaling defects combined with additional asymmetric variations in ligand recognition or constitutivity, demonstrating that the receptor is activated as a dimer through symmetrical, intermolecular conformational changes that culminate in trans-autophosphorylation. (iv) Continuing downstream, AgrC activation leads to the induction of the effector molecule RNAIII, whose mechanism of global gene regulation was unknown. We show that RNAIII functions by blocking the translation of a major pleiotropic transcription factor, Rot. (v) This work concludes with the construction of congenic S. aureus strains possessing divergent agr loci, which represent tools to understand the broader biological significance of agr specificity and cross-inhibition. |
