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Title: | Investigations into the Biochemical Mechanism of S. aureus agr Quorum Sensing Pheromone Recognition and Biosynthesis |
Authors: | Bodine, Steven Parker |
Advisors: | Muir, Tom W. |
Contributors: | Chemistry Department |
Keywords: | Bacterial pathogenesis Bacterial signaling Quorum sensing RiPP biosynthesis Staphylococcus aureus Synthetic biology |
Subjects: | Biochemistry Molecular biology Chemistry |
Issue Date: | 2024 |
Publisher: | Princeton, NJ : Princeton University |
Abstract: | In the opportunistic pathogen Staphylococcus aureus, a master regulator of pathogenicity is the accessory gene regulator (agr) quorum sensing circuit. Via the production and recognition of a secreted autoinducing peptide (AIP) signaling molecule, S. aureus communicates within its local environment to coordinate gene expression and behavior. Disruption of agr quorum sensing is sufficient to ablate S. aureus cytotoxin expression, making this quorum sensing system an attractive therapeutic target. One proposed mechanism to disrupt agr quorum sensing is the inhibition of AIP biosynthesis. The AIP is a ribosomally synthesized and post translationally modified peptide (RiPP) derived from the cleavage of a precursor peptide AgrD in a multistep peptidolytic cascade. The first cleavage event in this biosynthetic pathway is well studied; the integral membrane protease AgrB cleaves the C-terminal domain of AgrD and cyclizes the C-terminus of the cleavage product with an internal cysteine thiol to generate a macrocyclic biosynthetic intermediate. However, despite decades of research, the identity of the protease(s) responsible for the second step(s) of AIP maturation has not been confirmed. Recently, the integral membrane protease MroQ was discovered in S. aureus and shown to exhibit regulatory behavior over agr. We demonstrate that MroQ serves as the protease catalyzing the second proteolytic cleavage event of AIP biosynthesis. Genetic complementation reveals that MroQ proteolytic activity is necessary for AIP biosynthesis in multiple S. aureus agr variants, while in vitro biochemical experiments demonstrate that AgrD, AgrB, and MroQ are sufficient for AIP biosynthesis in agr specificity groups -I and -II. Furthermore, we elucidate the molecular determinants of MroQ cleavage-site recognition, which are critical to MroQ’s differential activity between distinct agr specificity groups. Altogether, this study deepens the collective understanding of agr quorum sensing and identifies a novel target for anti-virulence pharmaceutical development. |
URI: | http://arks.princeton.edu/ark:/88435/dsp01jq085p354 |
Type of Material: | Academic dissertations (Ph.D.) |
Language: | en |
Appears in Collections: | Chemistry |
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