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 ester-linked 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.

Figure 1

Chemical synthesis of AgrD thiolactone-containing autoinducing peptides via a solid-phase intramolecular chemical ligation strategy. Key to this process is the ability to prepare a fully unprotected peptide immobilized on a solid support through a reactive thiol ester bond. Simply swelling such an unprotected peptide-[thioester]-resin in aqueous buffer results in a chemoselective ligation reaction and concomitant cleavage of the peptide from the support. This final step is made possible because of the excellent swelling properties of the PEGA support in water (21).

Figure 2

Synthetic thiolactone peptides are biologically active. Shown are representative data for activation (A) and inhibition (B) of the agr response by a synthetic thiotactone peptide. Degree of activity based on β-lactamase activity shown as a plot of V_max versus peptide concentration. (A) Activation of the agr response in group II S. aureus cells by synthetic AgrDII. (B) Inhibition of the agr response in group I S. aureus cells by synthetic AgrDII.

Figure 3

Attenuation of the staphylococcal skin abscess by a synthetic thiolactone-containing peptide. (A) Effect of inhibitory peptide on murine skin abscesses. Six- to eight-wk-old hairless mice were injected s.c. in the flank area on day zero with approximately 10⁸ colony-forming units of S. aureus from an exponential-phase culture grown in CY broth. The size of the resulting s.c. abscesses were measured with dividers every 24 hr; sizes were calculated by assuming that lesions were approximately elliptical and of approximately uniform depth. Four mice were injected with strain RN6390B (agr+), four with RN6911 (agr-null), three with RN6390B + 5 μg of synthetic AIPII (group II thiolactone peptide), and three with RN6390B + 10 μg of synthetic AIPII. In each case the peptide was included with the bacteria being injected. The graph shows the data plotted as average lesion size, with error bars representing the SD. The differences among the sizes of the lesions induced by RN6911 and by RN6390B plus the peptide are not significant. (B–D) Typical lesion sizes at day 5 are shown. (B) RN6390B; (C) RN6911; (D) RN6390B + 5 μg of synthetic AIPII.

Figure 4

Effect of replacing each residue within the AgrDII peptide sequence with alanine on activation (A) and inhibition (B) activity. The results are superimposed on a model of the AgrDII peptide created by using the program discover (Biosym Technologies, San Diego). The ED₅₀ values (activation, group II cells) were as follows: Ala-1, 3.2 ± 2.2 nM; Ala-2, 73.7 ± 1.9 nM; Ala-3, no activity; Ala-6, 33.6 ± 1.5 nM; Ala-7, <1 nM; Ala-8, no activity; Ala-9, no activity. The IC₅₀ values (inhibition, group I cells) were as follows: Ala-1, <1 nM; Ala-2, <1 nM; Ala-3, <1 nM; Ala-6, ≪1 nM; Ala-7, <1 nM; Ala-8, no activity; Ala-9, no activity. None of the peptides activated the agr response in group I S.aureus strains or inhibited the agr response in group II strains.

Figure 5

Proposed model for the activation and the inhibition of the agr response. (A) Activation of the agr response occurs when an AgrD peptide interacts with an AgrC receptor from the same S. aureus class. Specific AgrD–AgrC interactions lead to the thiol ester group within the peptide being positioned close to a nucleophile within the receptor. Subsequent trans-acylation leads to a signal-transducing conformational change in the receptor, which may include homodimerization and trans-phosphorylation of the histidine kinase domains. (B) Inhibition of the agr response occurs via an interclass, noncovalent interaction that serves to exclude the strain’s own activating peptide from the receptor.