Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Anushree Chatterjee

Second Advisor

Prashant Nagpal

Third Advisor

Corrella Detweiler

Fourth Advisor

Robert T. Batey

Fifth Advisor

Stephanie J. Bryant

Abstract

Antibiotic resistance is a growing threat to global healthcare that requires immediate action to avoid the post-antibiotic era. The inherent ability of bacteria to obtain resistance and the lack of new antibiotics has led to the current antibiotic crisis. Current antibiotics are typically found through soil compound screens and only target proteins within three cellular pathways: cellular replication, cell wall biosynthesis, and protein biosynthesis. In the last decade, strains have been isolated which have resistance to nearly all available antibiotics highlighting the urgent need for intervention. In this work we investigated the rational design of non-naturally derived antibiotics which target bacterial processes outside of the three traditional antibiotic target pathways.

Antisense therapeutics are nucleic acid oligomers that bind sequence-specifically via Watson-Crick base pairing with native nucleic acids and inhibit translation of the targeted gene. For this work, we use non-natural nucleic acid analog oligomers, peptide nucleic acids (PNA), for their demonstrated intracellular stability and high binding affinity for native nucleic acids. In our initial study, we show PNA oligomers targeted to TEM-1 β-lactamase re-sensitized drug-resistant Escherichia coli to a β-lactam antibiotic. We further adapted E. coli to low levels of PNA and β-lactam antibiotic and observed high variability in expression of stress response genes possibly suggesting a bet-hedging type adaptive resistance. In our next study, we designed PNA to target essential bacterial genes in non-traditional antibiotic target pathways. We designed the PNA against the genome sequences of non-pathogenic, drug sensitive E. coli, Klebsiella pneumoniae, and Salmonella enterica and subsequently tested their antibacterial action in multidrug-resistant (MDR) clinical isolates of the same three species. We found that 54% of predicted targets were effective at inhibiting the MDR pathogens demonstrating the ability to design sequence-specific yet still broad-pathogen antisense therapeutics. We further demonstrated that combinations of these essential gene antisense PNA and small molecule antibiotics function synergistically to enhance bacterial inhibition despite the clinical strains high antibiotic resistance.

We next focused our efforts on designing an antimicrobial agent for perturbing bacterial redox homeostasis. Reactive oxygen species (ROS) have been studied for their effect on antibiotic efficacy and the emergence of drug resistance. In this work, we studied one ROS in particular, superoxide, for its role as an oxidative stress catalyst and its demonstrated disruption of metal homeostasis in bacteria. To controllably produce superoxide, we investigated the design of quantum dot nanoparticles. When quantum dots are excited over their nominal bandgap, excited electrons and holes are available for redox half reactions in the biological environment. In this work, we demonstrated the tuning of quantum dots for superoxide production from molecular oxygen and further showed the tuned nanoparticles inhibition of clinical isolates. Further, we established that E. coli could be eradicated from co-culture with mammalian cells; leaving the mammalian cells intact.

Given the role that ROS have been shown to have in bactericidal antibiotic efficacy, we hypothesized that our superoxide-producing nanoparticles would function synergistically with small molecule antibiotics. Indeed, our designed superoxide generating nanoparticles potentiated the activity of antibiotics in clinical MDR isolates in spite of their antibiotic resistance. In this study, superoxide potentiated the activity of bactericidal antibiotics as well as bacteriostatic antibiotics, which had not been shown previously. To better understand the effect of superoxide generation on bacteria, we performed analysis of E. coli’s transcriptome during treatment. We removed mate

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