Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence

Abstract

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specificity, S. aureus strains can be placed in at least four different groups A. Figuereido and R.P.N., unpublished data).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...

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Numerous SAR studies have mapped amino acid residues critical for AIP activity, and overall the results demonstrate that critical amino acids differ between AIP groups (218)(219)(220). For example, in AIP-I the endocyclic aspartic acid residue adjacent to the cysteine residue is critical, as replacement of this amino acid with alanine converts AIP-I to a potent inhibitor of AgrC of groups I and IV (218,219). In contrast, mutation of the exocyclic residue asparagine of AIP-II to alanine converted the peptide to a self-antagonist, while mutation of the endocyclic residues did not alter its activity (221).…”

“…The molecular determinants for AIP specificity have been shown to reside entirely in the interaction between the AIP signal and the AgrC receptor (130). Both cognate activation and heterologous inhibition activities require the ring structure of AIP, as linear AIPs have no detectable activity in any agr group (219,220), but the requirement of the thioester linkage has been debated (218)(219)(220). The tail of the AIP molecule is required only for activation, as mutation of the tail amino acid residues or removal of the tail did not alter inhibition of the agr response in heterologous groups despite eliminating self-activation activity (130,219).…”

“…The tail of the AIP molecule is required only for activation, as mutation of the tail amino acid residues or removal of the tail did not alter inhibition of the agr response in heterologous groups despite eliminating self-activation activity (130,219). Numerous SAR studies have mapped amino acid residues critical for AIP activity, and overall the results demonstrate that critical amino acids differ between AIP groups (218)(219)(220). For example, in AIP-I the endocyclic aspartic acid residue adjacent to the cysteine residue is critical, as replacement of this amino acid with alanine converts AIP-I to a potent inhibitor of AgrC of groups I and IV (218,219).…”

“…Both cognate activation and heterologous inhibition activities require the ring structure of AIP, as linear AIPs have no detectable activity in any agr group (219,220), but the requirement of the thioester linkage has been debated (218)(219)(220). The tail of the AIP molecule is required only for activation, as mutation of the tail amino acid residues or removal of the tail did not alter inhibition of the agr response in heterologous groups despite eliminating self-activation activity (130,219). Numerous SAR studies have mapped amino acid residues critical for AIP activity, and overall the results demonstrate that critical amino acids differ between AIP groups (218)(219)(220).…”

“…This may help to explain why residues critical for activity differ between AIP groups. Systematic mutation of native AIP molecules has generated analogues with various changes in activity, including reduced activation activity, enhanced activation activity, loss of all activity, conversion of self-activation to self-inhibition activity, and a unique case of conversion of cross-inhibition to cross-activation activity (218)(219)(220)(221). Combining multiple mutations known to provide antagonist activity, for example, truncating the tail of a selfinhibitory AIP-I mutant, has also been shown to have potential in the generation of stronger inhibitors that function against all agr groups (218).…”