Characterization of the Response of Pseudomonas Aeruginosa to the Novel Bactericidal Agent AB569 and its use as a Model Organism in Microbial Fuel Cells
Year: 2018
Type: article
Abstract: Cystic Fibrosis (CF) is a chromosomally recessive disorder characterized by a lack of functional CFTR transporter and an accumulation of mucus in the several organ systems, but perhaps most notably in the lungs.Here, resident mucus becomes thick and unable to be cleared by body naturally.This allows inhaled bacteria to thrive within the nutrient rich mucus, leading to infections that are hard to clear, often becoming chronic infections afflicting a patient for life.Traditional antibiotics are often useless against these chronic infections, often leading to patient suffering without any effective option.In 2006, our lab published that acidified nitrite could be used to combat mucoid Pseudomonas aeruginosa, a form often seen with chronic CF infections.This dissertation reports on the ability of the metal chelator, EDTA, to potentiate the action of nitrite even further.This activity was first discovered during an investigation on an integral hypothetical ABC transporter in P. aeruginosa, PA4455.Experiments related to this project showed that a deletion mutant of PA4455 was sensitive to both EDTA and acidified NaNO2, and exquisitely so to the combination.However, it also showed efficacy against wild-type P. aeruginosa PAO1.This was unexpected, and prompted an investigation into its use against other bacteria.This combination, termed AB569, does in fact show efficacy against a number of bacterial species, both Gram-negative and Gram-positive.Using a combination of broth based killing assays and FIC checkerboard analysis, we showed that AB569 is effective against a myriad of microbes, but was notably synergistic against Gram-negative organisms.Additionally, we performed biofilm killing studies using flow cytometry for endpoint analysis, showing that AB569 is able to kill approximately 70% of established biofilms of 2 of the ESKAPE pathogens, P. aeruginosa and S. aureus.To determine the underpinning mechanism of how AB569 was working, genetic and electrochemical approaches were taken.RNA-sequencing of AB569 III treated P. aeruginosa revealed that nearly 75% of genes deemed essential in its genome were downregulated.This included mechanisms for a number of housekeeping functions, including transcription/translation and DNA synthesis.Finally, we tested the safety and efficacy of AB569 on human cell lines and in a mouse chronic infection model.We found that monolayers of cells could be treated with bactericidal concentrations of AB569 without significant cell death and treatment of chronic infection revealed a reduction in CFU per lung, with no concomitant histopathological findings.Overall, we are pleased to report that AB569 offers a significant antimicrobial effect with low toxicity, potentially offering the ability for it to be used as a therapeutic.This dissertation also details an exploration of the fields of synthetic biology and microbial fuel cells (MFCs), and how specifically metabolic reengineering could be used to create organisms with exceptionally more electrogenic potential.We explore the possibility of bridging the urease activity of PAO1 with the ammonia oxidation of Nitrosomonas europaea, producing nitrite, another substrate usable by PAO1.This organism would be intensely more metabolically active than PAO1, a desirable feature for MFC bacteria. Table of ContentsList of Tables and Figures VII Chapter 1 -An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability 1 Chapter 2 -A Putative ABC Transporter Permease is Necessary for Resistance to Acidified Nitrite and EDTA in Pseudomonas aeruginosa under Aerobic and Anaerobic Planktonic and Biofilm Conditions 38 Chapter 3 -A Nontoxic Chemical Tandem That Kills All Bacteria 68 Chapter 4 -Exploring the Field of Synthetic Biology and A Potential Application in the Engineering of a More Electrogenic Pseudomonas aeruginosa
Author: McDaniel, Cameron T
Institution: University of Cincinnati
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7017930854