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Title: Rapid testing of antimycobacterial agents and identification of acid-regulated genes in Mycobacterium spp. using bioluminescence
Author: Shafi, Jamila
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2001
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The WHO has declared tuberculosis 'a global emergency'. New treatments are needed and bioluminescence was investigated in this regard. Bioluminescence has been demonstrated as an ideal tool for a reporter of bacterial viability, detection and gene expression. The first aim of the project was to develop a method for rapid screening of antimycobacterial drugs. Mycobacterium smegmatis and Mycobacterium aurum containing luxCDABE genes from Photorhabdus luminescens generated no light. Bioluminescent strains of M. bovis BCG and M. tuberculosis were generated although this property was unstable. Studies comparing lux genes from Vibrio harveyi and P. luminescens were done to explain these findings. Bioluminescence could be used to evaluate the activity of antimycobacterial agents against mycobacteria in macrophages using a luminometer and a CCD camera. A comparison of the alamar blue and bioluminescent assays for rapid estimation of antimycobacterial activity of drugs against recombinant mycobacteria was done. Overall, the bioluminescent assay offered the greatest potential for use in high-throughput screening of novel antimycobacterial agents against mycobacteria. The second aim of the project was to identify genes from M. tuberculosis regulated by acid and/or involved in the acid tolerance response (ATR). Bacterial lux was used to assay in vitro regulation of expression of two genes, Rv3521c (putative decarboxylase) and phoPR (two-component regulator). Induced lux expression downstream of Ppd and PphoPR promoters were seen in M. smegmatis demonstrating acid-inducibility, but no lux expression was seen in M. bovis BCG. An ATR was determined in M. smegmatis and BCG. A putative knockout mutation of Rv3521c was constructed in M. smegmatis. Acid tolerance was not exhibited by this mutant with phosphoric acid as the acidulant but was seen when hydrochloric acid, suggesting different mechanisms are employed to induce tolerance to different acidulants.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available