Student Theses and Dissertations

Date of Award

2015

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Muir Laboratory

Abstract

The accessory gene regulator (agr) locus in the commensal human pathogen, Staphylococcus aureus, is a two-promoter operon with allelic variability that encodes a quorum sensing circuit involved in regulating virulence in the bacterium. Secretion of unique autoinducing peptides (AIPs) and detection of their concentration via AgrC, a transmembrane receptor histidine kinase, coordinates local bacterial population density with global changes in gene expression. In order for S. aureus to produce AIP, three proteolytic transformations involving the proteins AgrB and SpsB are required. However, despite our current understanding of AIP peptide processing, the actual manner in which the AIP crosses the cellular membrane, and specifically the role of AgrB in this process, has not been examined. Neither is it clear whether SpsB cleaves all four variants of the AgrD precursor peptide. Initially, the aims of this thesis were to: 1) determine the role of AgrB in secreting the AgrD(1-32)-thiolactone and 2) confirm that SpsB is the protease responsible for the final cleavage step of AgrD in AIP biosynthesis. To achieve these goals, an in vivo secretion assay using intein chemistry to produce AIP in the absence of AgrB was developed to examine whether AgrB facilitates AIP secretion. Also, SpsB biochemical assays were used to provide a thorough investigation of the final cleavage step in AIP biosynthesis for AgrD-I and AgrD-II. The findings of this work indicate that AgrB does not facilitate secretion, and SpsB can only cleave AgrD-I correctly but not AgrD-II. Taken together, these observations suggest that AgrB and SpsB are two proteins associated with AIP biosynthesis, but there are likely other proteins that need to be identified. The final aim of this work was to investigate the effect of AIP macrocycle size on AgrC activation. Since staphylococcal virulence can be inhibited through antagonism of its quorum sensing system, there has been tremendous interest in understanding the structure-activity relationships underlying the AIP-AgrC interaction. The defining structural feature of the AIP is a 16-membered, thiolactone-containing macrocycle. However, the importance of ring size on agr activation or inhibition has not been explored. This deficiency is addressed through the synthesis and functional analysis of AIP analogs featuring enlarged and reduced macrocycles. This study is the first to interrogate AIP function using both established cell-based reporter gene assays and newly developed in vitro AgrC-I binding and autophosphorylation activity assays. Based on our data, we present a model for robust agr activation involving a cooperative, 3-points-ofcontact interaction between the AIP macrocycle and AgrC.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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