Date of Award

Spring 5-29-2025

Document Type

Thesis (Ph.D.)

Department or Program

Chemistry

First Advisor

F. Jon Kull

Second Advisor

Charles R. Midgett

Abstract

Antibiotic resistance has exacerbated the global health crisis of diarrheal diseases caused by enteric bacterial pathogens. Investigating pathogens, such as Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae (cholera), helps to uncover mechanisms for the inhibition of virulence gene expression. A notable member of the AraC transcription factor family in ETEC, Rns, regulates virulence factors including pili and fimbriae, which are essential for adherence and colonization of the intestinal epithelial layer. This microbe–host interaction initiates the release of toxins, leading to the water and ion loss in clinically diagnosed as diarrhea.

In the canonical N-terminal domain of Rns, mutagenesis of the binding pocket confirmed that residues R75 and H20 are crucial for coordinating the carboxylate head group of decanoic acid, an inhibitor of Rns activity. Pocket-filling mutations I14F and I17F reduced the pocket volume, blocking binding of the decanoic acid observed in the wild-type RnsWT. Additionally, disruption of the dimerization interface by mutagenizing Y139 prevented pi-pi stacking interactions with residue Y139 of a nearby protomer, resulting in monomeric RnsY139E in solution. The 2.1Å X-ray crystal structure of Rns reveals new interactions between residues E139 and K143 across the crystal lattice, giving rise to a dimer in the crystal. In RnsWT, however, K143 forms a salt bridge with the neighboring protomer residue E121.

Crystallographic studies probing the DNA-binding domain, in the presence of a CS3 promoter sequence containing sticky ends, produced an approximately 3.4Å diffracting crystal with a longer unit cell, consistent with other protein–DNA co–crystal unit cells, suggesting the possible presence of DNA. Preliminary analysis of the complex structure, solved by molecular replacement, suggests that DNA might be present.

Together, this body of work establishes key residues involved in Rns inhibition and advances understanding of the mechanistic inhibitory pathways of AraC–VRs.

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