Student Theses and Dissertations

Date of Award

2002

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Muir Laboratory

Abstract

Two-component signaling systems involving receptor-histidine kinases (RHKs) are present in all branches of life outside of the animal kingdom. Remarkably, very little is known concerning the extracellular ligands that presumably bind to R H K s to initiate signaling. The two-component agr signaling circuit in the bacterial pathogen, Staphylococcus aureus, is one system where the ligands are known in chemical detail. These ligands (the AIPs) are short (7-9 aminoacyl residue) peptides containing a thiolactone structure, in which the oc-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. In the agr system, the AIPs generally activate virulence expression in the producer strain and cross-inhibit virulence expression in S. aureus strains expressing other AIPs. In this study, genetic studies on the receptor-histidine kinase, AgrC, have demonstrated that intra-group activation and inter-group inhibition are both mediated by the same group-specific receptors. Pharmacological analysis has shown that the AIPs compete for a c o m m o n binding site on the receptor, and this site has been partly localized through the use of chimeric receptor analysis. Structure-activity relationship (SAR) studies have identified residues within the AIPs that are critical for receptor binding and activation. These results have facilitated the development of global inhibitors of virulence in S. aureus, which, in one case, consists of a truncated version of one of the naturally occurring AIPs. Cell-based in vitro assays of this peptide have demonstrated IC50's in the n.anomolar range for inhibition of the transcription of a known global regulator of virulence in S. aureus, RNAIII. Virulence inhibition in vivo has been seen with native AIPs in a murine subcutaneous abscess model. Ongoing studies are focused on peptidomimetic design to generate stable and pharmacologically relevant lead compounds. These results collectively suggest that the design of molecules that compete with natural agonists for binding at R H K sensor domains could represent a general approach to the inhibition of R H K signaling.

Comments

Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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