The synthesis of virulence factors and other extracellular proteins responsible for pathogenicity in Staphylococcus aureus is under the control of the agr locus. A secreted agr-encoded peptide, AgrD, processed from the AgrD gene product, is known to be an effector of self-strain activation and cross-strain inhibition of the agr response. Biochemical analysis of AgrD peptides isolated from culture supernatants has suggested that they contain an unusual thiol esterlinked cyclic structure. In the present work, chemical synthesis is used to confirm that the mature AgrD peptides contain a thiolactone structure and that this feature is absolutely necessary for full biological activity. The AgrD synthetic thiolactone peptides exhibited biological activity in vivo in a mouse protection test. Structure-activity studies have allowed key aspects of the peptide structure involved in the differential activation and inhibition functions to be identified. Accordingly, we propose a model for activation and inhibition of the agr response in which the former, but not the latter, involves specific acylation of the agr transmembrane receptor, AgrC.Staphylococcus aureus is among the major nosocomial pathogens that are now under increasing risk of developing resistance to all currently available therapeutics. Consequently, there is a pressing need to identify new types of antibacterial agents, and it has been suggested that interference with the expression of virulence may represent a promising antibacterial modality (1, 2). As it happens, S. aureus is a very good candidate for such an approach as it uses a global regulator, agr, activated by secreted autoinducing peptides, to control the expression of most virulence genes (3, 4). These peptides serve as ligands for a signal receptor, AgrC, that initiates this agr response through a classical two-component signaling pathway. The autoinducer-receptor pair shows considerable interstrain sequence variation, which affects the specificity of the receptor-ligand interaction. On the basis of this autoinducerreceptor specificity, S. aureus strains can be placed in at least four different groups A. Figuereido and R.P.N., unpublished data). Remarkably, each of the secreted autoinducing peptides can activate the agr response within the same group and inhibit the agr response in strains belonging to the other groups. This inhibition constitutes a novel form of bacterial interference (5) because it affects the expression of a specific group of genes rather than inhibiting growth, which is the usual manifestation of bacterial interference.The agr locus contains two divergent promoters, P2 and P3. There are four genes, (agrA-D) in the P2 operon that code for the cytosolic, transmembrane, and extracellular components of a density-sensing͞autoinduction circuit (4). The agrD gene product is a propeptide that is processed and secreted through AgrB, an integral membrane protein. The resultant mature autoinducing peptide (AIP) is thought to bind to the transmembrane receptor coded by AgrC. Bind...

Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure-activity relationship studies. These ligands are short (8-to 9-aa) peptides containing a thiolactone structure, in which the ␣-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

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