Tuberculosis (TB) caused by the Mycobacterium tuberculosis complex (MTBC) remains one of the most important infectious diseases of mankind. Isoniazid (INH) and rifampicin are the main first line drugs used in multi-drug treatment of TB. However, the necessary duration of treatment with these drugs is impractically long and development of resistance against these compounds and other TB drugs is an increasing impediment to treatment programs. As a result, there is a requirement for research and development of new TB drugs which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as INH and rifampicin.

The overall aim of this project was to utilise transposon sequencing genetic screens (TnSeq) of M. bovis BCG to identify novel drug targets that could potentiate the activity of INH and rifampicin and be developed as new multidrug therapies. In work leading up to this project, TnSeq had been used to identify mutants with enhanced sensitivity to a sub-MIC concentration of INH. In the present project, a subset of these gene mutants that are putatively related to oxidative stress/redox reactions, namely bpoB, ephB and bcpB, were selected for validation by performing minimum inhibitory concentrations (MIC) and comparing to wild-type and genetically complemented mutants. KatG activity and transcription was measured with a catalase assay and quantitative PCR to test the hypothesis that absence of bpoB, ephB or bcpB leads to an increase in KatG levels, which in turn leads to an increased activation of the drug INH and thus increased sensitivity to INH. However, in this project we did not find any significant difference in the sensitivity pattern to INH or in KatG expression between the WT, gene mutants and complemented strains.

This project also described a Tnseq analysis to identify mutants with enhanced sensitivity to a sub-MIC level of rifampicin. The putatively rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups : firstly, genes that were involved in DNA/RNA metabolism, secondly, genes that were transporters and potentially regulating the export of drugs from the mycobacterial cell and lastly a group of genes that were identified as transcriptional regulators and kinases and has the ability to regulate the cellular systems in the mycobacteria. The gene-set includes several genes with established connections to drug/rifampicin sensitivity, thus validating the selection. Future work is needed to characterise the mechanisms of action of these gene-drug interactions and how they might be developed as potential candidate drug targets against Mtb. together with INH and rifampicin.