Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

First Advisor's Name

Yuk-Ching Tse Dinh

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Yuan Liu

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Fenfei Leng

Third Advisor's Committee Title

committee member

Fourth Advisor's Name

Lou Kim

Fourth Advisor's Committee Title

committee member

Keywords

Tuberculosis, Topoisomerase, Mycobacteria, Toxin, TB therapy, Ethacridine, Resistance, Inhibitors, Mutants, WGS

Date of Defense

11-2-2018

Abstract

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a global health problem for years. The emergence of drug resistance in this organism generates the necessity of exploring novel targets and developing new drugs. Topoisomerases are enzymes found in all kingdoms of life responsible for overcoming the topological barriers encountered during essential cellular processes. The genomes of mycobacteria encode only one type IA topoisomerase (MtopI), which has been validated as a novel TB drug target. The goal of this study is to obtain new information on the mechanism and resistance of endogenous and synthetic inhibitors of MtopI.

Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the toxin), with endoribonuclease activity. Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C-terminal domain of MtopI for mutual inhibition. In this study the interaction of Rv1495 with the positively charged C-terminal tail in Mtop I is reported. This new information is useful for rational design and discovery of antibiotics for mycobacteria.

Ethacridine, an FDA approved drug has shown activity against MtopI. In this project we studied the mechanisms of resistance associated with this drug as well the use of Ethacridine in combination with Moxifloxacin, to potentiate the bactericidal effect of this current second line drug for TB treatment. Results from sequencing of the genomic DNA isolated from the resistant mutants suggested the involvement of the Holliday-junction Ruv resolvase. Further studies showed that co-treatment with Ethacridine can enhance the moxifloxacin-mediated killing of M. smegmatis.

FP-11g, a novel fluoroquinophenoxazine inhibitor of bacterial topoisomerase I, has shown promising activity against M, tuberculosis. We explored the bactericidal activity and resistance mechanisms associated to FP-11g using M. smegmatis as model organism. Additionally, the inhibitory effect of FP-11g was also evaluated on M. abscessus. FP-11g at concentration 4X MIC showed complete bactericidal activity against M. smegmatis after 24 hours. Inhibitory activity against M. abscessus was also observed. Results from sequencing of the genomic DNA isolated from the M. smegmatis resistant mutants revealed mutations in genes associated with general drug resistance.

Identifier

FIDC007014

Available for download on Friday, October 18, 2019

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