Investigating the mode of action of tuberculosis drugs using hypersensitive mutants of Mycobacterium smegmatis by Richard Laurence Campen BSc(Hons.) Victoria University of Wellington A thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Microbiology Victoria University of Wellington 2015 Primary Supervisor Dr Ronan O’Toole Senior Lecturer, School of Medicine University of Tasmania Victoria Supervisor Professor John H. Miller School of Biological Sciences Victoria University of Wellington Abstract Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is the leading cause of death and disease by a bacterial pathogen worldwide. The growing incidence of drug resistant TB, especially multi-drug resistant TB highlights the need for new drugs with novel modes of action. Current treatment of TB involves a multi-drug regimen of four drugs including isoniazid and rifampicin, both of which were discovered over 40 years ago. Bedaquiline is one of the first novel TB drugs to enter clinical trials since the discovery of rifampicin, and has shown excellent activity against drug resistant TB. Although isoniazid and rifampicin are well established anti-TB drugs, significant gaps in knowledge regarding their modes of action exist. Furthermore, little information on the mode of action of the novel drug bedaquiline is known beyond its primary target. Characterisation of drug mode of action facilitates rational modifications of drugs to improve the treatment of TB. The aim of this study was to identify novel aspects of the modes of action of isoniazid, rifampicin, and bedaquiline by characterising drug hypersensitive mutants of M. smegmatis mc2155. A sub-saturated M. smegmatis mc2155 transposon mutant collection with 1.1-fold genome coverage (7680 mutants) was constructed, with this collection estimated to contain mutations in 73.2% of all genes capable of maintaining a transposon insertion (non-essential genes). A high-throughput assay was developed for screening the collection, and mutants related to known drug mode of action were identified for isoniazid (ahpC and eccCa1) and bedaquiline (atpB). Additionally, known mechanisms of drug inactivation were identified for isoniazid (nudC), rifampicin (arr and lspA), and bedaquiline (mmpL5). The finding that transposon mutants of nudC are hypersensitive to isoniazid independently validated the recent discovery of the role of NudC in basal isoniazid resistance by Wang et al. (2011). The remaining genes identified in this thesis represent potentially novel aspects of the modes of action or resistance mechanisms of these drugs. Cross-sensitivity to other drugs indicated that the mechanism of sensitivity was drug specific for the mutants examined. Differential-sensitivity testing against drug analogues revealed that Arr is involved in resistance to the rifampicin analogue rifapentine as well, indicating that Arr can detoxify rifapentine similar to rifampicin. The nudC mutant showed increased sensitivity to a range of isoniazid analogues, indicating that it can detoxify these analogues i similar to the parent compound. Interestingly six analogues were found to be less active against the nudC mutant than expected. A number of overexpression strains were tested against these six analogues; a nudC overexpression strain, and a strain overexpressing inhA, the primary target for isoniazid. Overexpression of nudC as well inhA increased the resistance of WT to isoniazid, but failed to increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. Isoniazid is a prodrug and is activated by the peroxidase/catalase enzyme KatG. Overexpression of katG resulted in increased isoniazid sensitivity, as well as increased sensitivity to NSC27607, NSC33759, and NSC40350. Together these results suggest that NSC27607, NSC33759, and NSC40350 are activated by KatG, but that InhA is not the primary target. Additionally the inability of NudC overexpression to confer resistance suggests these analogues are not acting via a NAD adduct, the mechanism by which isoniazid inhibits InhA. These results suggest that there are other toxic metabolites being produced by KatG activation of these three analogues. In conclusion, characterisation of mutants identified in a high-throughput assay for drug hypersensitivity identified genes involved in the modes of action or resistance mechanisms for isoniazid, rifampicin, and bedaquiline. Additionally, a number of novel genes were identified that have no known connections to the known modes of action or resistance mechanisms for these drugs. Further testing of a nudC mutant revealed three isoniazid analogues that appear to inhibit growth of M. smegmatis mc2155 independent of InhA, the primary target of isoniazid. This study has successfully demonstrated that screening for drug hypersensitivity can generate novel information on drug mode of action and resistance mechanisms. This information can ultimately be used to help drive the development of new drugs, and improve treatment of TB. ii Acknowledgements First I would like to thank my primary supervisor Ronan O’Toole for taking me on as a PhD student, and thank you for your support and guidance throughout my project. I would also like to thank my Victoria supervisor Professor John Miller for his support during the later stages of my PhD project, particularly with proof reading my thesis. My gratitude also goes to Associate Professor David Ackerley for taking me in during the later stages of my PhD. Inclusion in your lab group helped me get through the last few years of my project. Thank you also for your help with proof reading my thesis. Thank you also to Dr Joanna Mackichan for sharing her lab space during the last few years of my PhD. I am also grateful to the fellow O’Toole lab students over the years, Shahista Nisa, Chris Miller, Nathaniel Dasyam, Ian Bassett, Mudassar Altaf, and Sandi Dempsy. Your help and guidance over the years was invaluable. My gratitude also to Yee Suen Low for her help with practical aspects of my project. Thank you also to the Ackerley lab group, including long time office mates Mark Calcott, Katherine Robins, and Becky Edgar, as well as the rest of KK815/816 for their support and friendship throughout my PhD. Thank you also to our collaborator Professor Greg Cook at the University of Otago, and his post-doc Jen Robson. Thank you for providing a sample of bedaquiline for this study, and for your help with the practical aspects of the bedaquiline assay. Thank you also for flying me down to Otago to discuss my project, and present my work to your research group. Thank you to my wonderful family and friends for all their support throughout my PhD. Especially thank you to my mother and father for their emotional and financial support, and to my father for sharing his roof with us for the last couple years. Finally, thank you to my amazing wife Kelly. Thank you so much for your love and support, it has been instrumental in me completing this project. iii iv Table of Contents Abstract ...................................................................................................................... i Acknowledgements ................................................................................................... iii Table of Contents ........................................................................................................ v List of Figures ............................................................................................................. xi List of Tables ............................................................................................................ xiii List of Abbreviations ................................................................................................ xiv 1. General Introduction .......................................................................................... 1 1.1. Tuberculosis ................................................................................................................. 1 1.1.1. Tuberculosis in New Zealand ............................................................................... 4 1.2. Pathogenesis ................................................................................................................ 4 1.3. Mycobacteria ............................................................................................................... 6 1.3.1. Physiology ............................................................................................................. 6 1.3.2. Mycobacterium tuberculosis ................................................................................ 8 1.3.3. Mycobacterium smegmatis .................................................................................. 9 1.4. Treatment of tuberculosis ......................................................................................... 10 1.4.1. Anti-tuberculosis Drugs ...................................................................................... 11 1.4.2. Drug resistance ................................................................................................... 13 1.4.3. Modes of action of the anti-tuberculosis drugs to be examined in this study .. 13 1.5. Drug Discovery ........................................................................................................... 15 1.5.1. Mode of action identification ............................................................................. 16 1.5.1.1.