<p>Tuberculosis continues to be a major world health problem, causing more deaths than any other bacterial disease. Long treatment durations using a complex cocktail of drugs are often associated with patient non-adherence to therapy, and this has accelerated the development of drug resistant strains. Tuberculosis drug resistance has developed to the extent that some strains are resistant to all clinically used drugs. Therefore novel tuberculosis treatment drugs are urgently required to combat these resistant strains, sterilise latent infections and reduce lengthy treatment durations. This research developed and optimised a high-throughput assay to screen chemical libraries for compounds with anti-mycobacterial activity. The assay utilised fast growing tuberculosis model species M. smegmatis expressing foreign green fluorescent protein (GFP). GFP allowed bacterial growth inhibition to be measured both by fluorescence in addition to absorbance. The assay was expanded to four different culture conditions two of which were nutrient starvation that better mimicked environmental conditions M. tuberculosis is exposed to during infection. These differential culture conditions also revealed previously unidentified mycobacterial inhibitors. Three chemical libraries totaling over 5,000 compounds were screened in the different culture conditions. Seleno-amino acids (Se-AAs), a novel class of anti-tuberculosis compounds, were discovered through screens in nutrient starvation conditions. Based on traits of strong inhibitory activity towards mycobacteria, low human cell line cytotoxicity, structural novelty and known over-the-counter sale as a non-prescription dietary supplement, the Se-AAs were chosen as a promising pharmacophore for further study. Using evidence derived from anti-sense gene knockdown, transposon mutagenesis and biochemical enzyme assays, a pro-drug hypothesis of anti-mycobacterial activity was proposed that involved Se-AAs being transported into the mycobacterial cell by nutrient uptake transporters and subsequent cleavage into catalytically active methylselenium species by lyase enzymes used in mycobacterial sulphurous amino acid metabolism. The activated methylselenium is reduced by mycobacterial redox homeostasis enzymes involved in mycobacterial oxidative defence such as alkyl hydroperoxidases, generating reactive oxygen radical products that damage mycobacterial DNA, lipids and proteins. Reduced methylselenol can be cycled back to the oxidised state by cellular mycothiones, continuously generating damaging reactive oxygen species within the mycobacterial cell. Methylselenium species also disrupt essential mycobacterial processes, such as ketosteroid catabolism and iron-sulphur cluster protein function. In summary, this research has designed and implemented a novel dual label differential culture condition assay useful in the screening and detection of chemicals with anti-tuberculosis properties. A novel structural class of anti-tuberculosis compounds with therapeutic potential, the Se-AAs, was discovered using this assay, the structure-activity relationship of the Se-AAs was explored and a three-component model of Se-AA anti-tuberculosis activity is proposed.</p>
Antibiotic action revealed by real-time imaging of the mycobacterial membrane
