Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated upon binding of a self-coded autoinducing peptide (AIP) to the receptor-histidine kinase, AgrC. The AIPs consist of a thiolactone macrocyle and an exocyclic "tail", both of which are important for function. In this report, characterization of the unique AIPs from the four known agr specificity groups of Staphylococcus aureus has been completed, along with analysis of cross-group inhibition of AgrC activation by each of the four AIPs. The following conclusions have been drawn: (i) The native thiolactone macrocyle and tail are necessary and sufficient for full activation by the AIPs, whereas the AIP-I macrocycle alone is a partial agonist. (ii) The native N-terminus is less critical, as that of AIP-I can be modified without affecting bioactivity, although that of AIP-III cannot. (iii) The ring and tail may function differently in different AIPs. Thus the group I and IV AIPs differ at a single (endocyclic) residue, which is the determinant of AIP specificity for these two groups and is essential for function. A similarly critical residue in AIP-II, however, is exocyclic. (iv) Cross-inhibition is more tolerant of sequence and structural diversity than is activation, suggesting that the AIPs interact differently with cognate than with heterologous receptors. (v) Chimeric peptides, in which the tails and macrocycles are switched, do not activate and instead inhibit receptor activation. These data suggest a model in which activation and inhibition involves different binding orientations within the ligand binding pocket of each receptor.

The staphylococcal virulon is controlled largely by the agr locus, a global accessory gene regulator that is autoinduced by a self-coded peptide (AIP) and is therefore a quorum sensor. The agr locus has diverged within and between species, giving rise to AIP variants that inhibit heterologous agr activation, an effect with therapeutic potential against Staphylococcus aureus: a single dose of an inhibitory AIP blocks the formation of an experimental murine abscess. As the AIP is unstable at physiological pH, owing to its essential thiolactone bond, its single-dose efficacy seems paradoxical, which has led us to analyze the in vivo kinetics of agr activation and the consequences of its blockage by a heterologous AIP. Initially, the infecting bacteria grow rapidly, achieving sufficient population density within the first 3 h to activate agr, and then enter a neutrophil-induced metabolic eclipse lasting for 2-3 d, followed by agr reactivation concomitantly with the development of the abscess. The inhibitory AIP prevents agr expression only during its short in vivo lifetime, suggesting that the agr-induced and therefore quorum-dependent synthesis of virulence factors shortly after infection is necessary for the subsequent development of the abscess lesion and bacterial survival. We confirm this finding by showing that a sterile agr ؉ supernatant causes a sterile abscess similar to the septic abscess caused by live bacteria. These results may provide a biological rationale for regulation of virulence factor expression by quorum sensing rather than by response to specific host signals.bioluminescent imaging ͉ in vivo gene expression ͉ bacterial pathogenesis A lthough there is a large body of information on the in vitro regulation of bacterial accessory genes involved in pathogenesis (referred to here as the virulon), the in vitro environment is highly artificial and applies only imperfectly, at best, to the real-life situation. To date, however, most in vivo approaches have generated single-point transcription profiles for infecting organisms but only a handful have addressed the expression pattern of virulence determinants through time, a critical feature of any infection (1 Agr is a complex locus that controls expression of a substantial part of the staphylococcal virulon (3, 4) consistent with its central role in pathogenesis (5-8). It consists of two divergent transcription units, driven by promoters P2 and P3 (9). The P2 operon contains four genes, agrB, agrD, agrC, and agrA, all of which are required for transcriptional activation of the agr system. AgrC is the receptor and AgrA the response regulator of a two-component signal transduction module that is autoinduced by a posttranslationally modified small peptide (AIP) (10, 11), processed by AgrB from the 46-residue agrD propeptide (12). The primary function of this four-gene unit is to activate the two major agr promoters, P2 and P3, significantly aided by a second regulatory protein, SarA (13, 14). The actual effector of agrdependent exoprotein gene regulation,...

Staphylococcal pathogenesis is regulated by a twocomponent quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling. Receptor-histidine kinases (RHKs)1 have been extensively characterized in bacteria and are present in archaea, microbial eukarya, and higher plants, where they have recently been shown (in Arabidopsis) to play a role in hormone signaling (1). In bacteria, RHKs are involved in sensing the environmental surroundings. Many of these kinases contain two transmembrane helices flanking a periplasmic domain. This domain contains the binding site for the appropriate ligand, such as metal ions (Mg 2ϩ in the case of PhoP) (2). Many RHKs have been shown to exist as preformed dimers in the inner cell membrane of Gram-negative bacteria (3). In the case of EnvZ, a pair of cytoplasmic ␣-helices from each monomer form a four-helix bundle (4). In Gram-positive bacteria, pheromone-inducible RHKs usually have a polytopic sensor domain, containing 5-8 membrane-spanning segments. RHKs predicted to possess this polytopic sensor domain include AgrC from Staphylococcus aureus (5), ComD from Streptococcus pneumoniae (6), ComP from Bacillus subtilis (7), and SapK from Lactobacillus sakei Lb706 (8). These RHKs respond to secreted signaling peptides, which bind to the sensor domain to initiate the transmembrane signal that activates the intracellular histidine kinase (HK). Notably, there is at least one RHK in Escherichia coli, UhpB, which contains 6 -8 transmembrane helices and which is also thought to function as a homodimeric complex (9).The agr system in S. aureus is the most fully characterized in terms of the receptor-ligand interaction. Density-dependent accumulation of an extracellular peptide, known as the AIP and derived from processing of the prepeptide, AgrD, triggers activation of the receptor-histidine kinase, AgrC. This leads to a downstream virulence response via the unique regulator, RNAIII (10). This signaling process is one example of densitydependent or "quorum-sensing" systems widespread in bacteria (11,12). The sequence of the AIPs is highly variable, resulting in at least four specificity groups within S. aureus and many others in other staphylococcal species (13-15). All strains within a group produce the same AIP. The agrB...

agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferasebased agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.The agr operon encodes a global regulatory system in the staphylococci, central to the biology of the organism (reviewed by Novick) (36). It controls a large set of genes, including most of those encoding extracellular virulence factors and many others encoding cytoplasmic proteins with catabolic and other functions. agr is highly conserved throughout the staphylococci but has diverged in a way that closely parallels speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that may represent the selective forces that have driven its evolution.agr operates by a classical two-component signaling module (Fig. 1A). It is autoinduced by a peptide, encoded within the locus, that is the ligand for the histidine kinase component of the signaling module. agr is therefore a sensor of population density or a quorum sensor. The agr variants represent specificity groups that determine the response to cognate or heterologous autoinducing peptides (AIPs). Although an AIP always activates its cognate agr locus, it competitively crossinhibits agr activation in most heterologous combinations. This cross-inhibition results in a novel type of bacterial interference in which the expression of accessory genes, but not growth, is blocked. This interference has potential therapeutic implications that are presently under investigation. In Staphylococcus aureus there are four known agr specificity groups, characterized by major sequence variations in a central region of the locus that encodes the AIP, the enzyme that processes it, and the receptor domain of the histidine kinase (7,19,21). The sequences flanking this central variab...