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Developing a Cell Based Screen for Inhibitors of Two Component Signal Transduction in Mycobacteria

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posted on 10.11.2021, 01:26 authored by Dempsey, Sandi Grainne

The growing number of drug resistant strains of Mycobacterium tuberculosis appearing worldwide has had an enormous impact on the ability to control and treat Tuberculosis (TB). Discovering new anti-TB drugs is of paramount importance to the global effort for TB eradication. The success of the pathogen is largely due to its inherent ability to remain in a non-replicating or latent state for extended periods of time. In order to achieve this shift it requires tightly controlled signal transduction mechanisms to respond to its host environment. Two component systems (TCS) are one example of signalling mechanisms employed by prokaryotes and are ideal candidates for antibacterial drug targets. It is understood that many TCS are conserved in a large number of organisms, they are often essential to the virulence and persistence of pathogens and they are virtually exclusive to prokaryotes. In this study three Mycobacterium smegmatis TCS were selected; DevS/DevR, MtrB/MtrA and SenX3/RegX3. Promoters under the control of these systems were cloned into an optimised mycobacterial high copy number GFP reporter plasmid and subject to a number of in vitro stress conditions to ascertain induction conditions for these systems. As expected the DevS/DevR controlled hspX promoter was responsive to oxygen starvation and the SenX3/RegX3 controlled phoA was induced by phosphate starvation. Interestingly, phoA and mtrA were also induced by magnesium chelator EDTA in minimal media. The phoA and mtrA promoter constructs were then used for in vitro high throughput bioassays with a number of compound libraries in order to screen for any inhibitory activity on each of the target systems. A phosphorylation inhibitor included in one of the screens, oleic acid, indicated that this assay could potentially be used to screen for TCS inhibitors, but no novel compounds were found in this study. As a proof of principle, known TCS inhibitors palmitoleic and oleic acid were employed to show a dose dependent inhibition mtrA expression. This method could potentially be expanded to other TCS of Mycobacterium smegmatis and Mycobacterium bovis BCG, or other signal transduction systems such as one component regulators and serine threonine kinases.

History

Copyright Date

01/01/2009

Date of Award

01/01/2009

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Cell and Molecular Bioscience

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Science

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

O'Toole, Ronan