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"MECHANISM OF QUINOLONE-MEDIATED LETHALITY"

by
Gan Luan
Molecular Biology, Genetics & Cancer Track
B.S. 2012, University of Science and Technology of China


Thesis Advisors: Xilin Zhao, Ph.D., Associate Professor
Karl Drlica, Ph.D., Professor
Public Health Research Institute and Department of Microbiology and Molecular Genetics

Tuesday, September 12, 2017
10:00 A.M., ICPH Auditorium


Abstract

The quinolones are lethal, clinically important antimicrobials whose activity is being eroded by bacterial resistance. These compounds act by trapping type II DNA topoisomerases on DNA as drug-enzyme-DNA complexes, called cleaved complexes, in which the DNA is broken. DNA breaks released from cleaved complexes cause chromosome fragmentation and cell death by two pathways, one that involves reactive oxygen species (ROS) and one that does not. My goal is to develop new quinolone derivatives that can bypass resistance and kill bacteria more effectively. Three aspects of quinolone action were studied. The first focused on cleaved complexes. The drug-magnesium-water bridge formed between fluoroquinolone and topoisomerase was shown to facilitate the formation and stabilization of cleaved complexes. Also, crosslinking with a ciprofloxacin derivative (Cip-AcCl) indicated the existence of two quinolone binding modes. The second focused on the paradoxical absence of killing at very high quinolone concentration. High, non-lethal concentrations of nalidixic acid induced lower levels of ROS, and lesions needed for ROS-mediated killing persisted at high quinolone concentration. Although protein synthesis inhibitors can block nalidixic acid lethality, experiments with a katG (catalase) deficiency suggests that inhibition of ROS accumulation, rather than protein synthesis inhibition, is the actual cause of paradoxical tolerance. The third part of the study focused on a novel role of RecF in quinolone-mediated lethality. A deficiency of RecF exhibited a protective effect on lethality caused by various quinolones, and this RecF effect is independent of the RecF recombinational DNA repair pathway. RecF stimulated both pathways used by quinolones to kill cells. Fewer double-strand DNA breaks were detected in a RecF deletion mutant than in wild-type cells when both strains were treated with ciprofloxacin, suggesting that RecF-mediated stimulation of quinolone lethality involves DNA breakage. Finally, an approach was developed to study additional proteins involved in RecF-mediated stimulation of quinolone lethality. Overall, the findings are expected to contribute to the development of new quinolone derivatives and to increase quinolone-mediated lethality.


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