CHARACTERISATION OF DRUG INDUCED CELL DEATH IN MYCOBACTERIUM SMEGMATIS Varsha Srivatsan A0082403 A thesis submitted for the degree in Master of Science Department of Microbiology National University of Singapore 2012 i Acknowledgements I am, most of all, very grateful to my supervisor, A/Prof Thomas Dick, for his guidance, advice and encouragement over the past year My sincere gratitude extends to Dr Paul Hutchinson and Mr Guo Hui from the flow lab who have been extremely helpful, kind, encouraging and supportive It their guidance that has helped me most of the analysis for my project I am thankful to Prof Sebastein Gagneux, my co-supervisor from Swiss TPH, Basel, Switzerland for his encouragement Doing my project would not have been so much fun if not for the members of the Drug discovery laboratory Ms Pooja Gopal and Mr Jian Liang from the DDL get a special mention for their patience, support and kind words of encouragement in every single step of the way I would also like to thank all the people at MD4 and MD4A who have helped me during the course of my project I would specially like to thank my dear friend, Ms Neetash M.R, for taking the interest to read my first draft and for giving her invaluable suggestions Lastly, and in no way the least, I am extremely grateful to my family and friends for just being there ii TABLE OF CONTENTS ABSTRACT v LIST OF TABLES vi LIST OF FIGURES vii INTRODUCTION 1.1 CELLULAR ENVIRONMENT INDUCED DEATH 1.2 ANTIBIOTIC INDUCED CELL DEATH 1.3 THE COMMON DEATH PATHWAY- GOING AGAINST THE DOGMA 1.4 MYCOBACTERIA- DO THEY SHOW THE COMMON PATHWAY? 13 20 MATERIALS AND METHODS 24 2.1 PREPARATION OF BASIC REQUIREMENTS (GROWTH MEDIA) 24 2.2 BACTERIAL CULTURES AND GROWTH VIABILITY MEASUREMENTS 24 2.3 DETERMINATION OF MIC/MBC OF ANTIBIOTICS 25  2.4 FLUORESCENCE ASSAY: MEASURING OH USING FLOW CYTOMETRY 27 RESULTS 29 3.1 DETERMINING GROWTH CHARACTERISTICS OF M.SMEGMATIS 29 3.2 MIC AND MBC OF ANTIBIOTICS 30 3.3 FLOW CYTOMETRY TO DETECT HYDROXYL RADICALS 31 DISCUSSION 47 CONCLUSION 56 FUTURE WORK 57 REFERENCES 58 APPENDIX 67 iv Abstract The study on Gram- positive and Gram-negative bacteria by Kohanksi et al (2007) challenged the longstanding notion about antibiotic induced cell death The authors showed that these bacteria illustrate a common death pathway when exposed to fluoroquinolones, aminoglycosides and β-lactams Cell death, they show, is caused by the production of hydroxyl radicals via the fenton reaction To determine whether the same pathway is triggered in the evolutionary distinct Mycobacteria, studies were conducted with the fast growing workhorse model organism, M smegmatis Upon adapting the protocol described in Kohanski (2008), it was found that we could measure hydroxyl radicals in M smegmatis generated by H2O2 We show that fluoroquinolones produce hydroxyl radicals, and that the quenching of these radicals resulted in increased bacterial survival indicating their involvement in causing cell death Three other drugs which are used in Tb therapy, isoniazid, kanamycin and ethambutol were also tested However, none of them significantly induced hydroxyl radicals These results suggest that, in contrast to other bacteria, mycobacteria harbor hydroxyl radical dependent as well as independent cell death pathways This study serves as a foundation to further our knowledge about pathways triggering reactive oxygen species and how we could use them to identify new targets for drug discovery If such a pathway is activated in M tuberculosis, it might prove to be very useful in our fight against drug resistant tuberculosis v List of Tables: Table 1: Minimum inhibitory concentrations and minimum bactericidal con- centrations of antibiotics used in the study v 30 List of figures Figure 1: Generation of superoxides and peroxides during respiration Figure 2: The proposed common pathway _ 15 Figure 3: Growth curve of Mycobacterium smegmatis _ 29 Figure 4: Assessment of hydroxyl radicals at hours and hours post H2O2 treatment 34 Figure 5: Fenton inhibition at hours post treatment with 20mM H2O2 with corresponding _ 34 Figure 6: Assessment of hydroxyl radicals at hours post ciprofloxacin treatment with the corresponding CFU _ 37 Figure 7: Assessment of hydroxyl radicals at hours post ciprofloxacin treatment with the corresponding CFU 37 Figure 8: Assessment of hydroxyl radicals at 24 hours post drug application with the corresponding CFU _ 38 Figure 9: Fenton inhibition at 24 hours post treatment with 0.8µM ciprofloxacin with the corresponding CFU 38 Figure 10: Assessment of hydroxyl radicals at 9h post treatment with MIC concentrations of levofloxacin, sparfloxacin and ofloxacin along with the corresponding CFU 39 Figure 11: Assessment of hydroxyl radicals at 24h post treatment with MIC concentrations of levofloxacin, sparfloxacin and ofloxacin along with the corresponding CFU 40 Figure 12: RFI (%) and the data representing the difference between the stained and unstained fluorescence values for fluoroquinolones 40 Figure 13: Isoniazid: RFI (%), data representing the difference between the stained and unstained fluorescence values and corresponding the CFU 42 Figure 14: Kanamycin: RFI (%), data representing the difference between the stained and unstained fluorescence values and corresponding the CFU 45 Figure 15:Ethambutol: RFI (%), data representing the difference between the stained and unstained fluorescence values and corresponding the CFU 46 vi Figure 16: Replication of Kohanksy (2007) E coli treated ofloxacin _ 67 Figure 17: Replication of Kohansky (2007) E coli treated kanamycin 68 vii Introduction The number of these Animals in the scurf of a man’s teeth are so many that I believe they exceed the number of Men in a kingdom For upon the examination of a small parcel of it, no thicker than a Horse-hair, I found too many living Animals therein, that I guess there might have been 1000 in a quantity of matter no bigger than the 1/100 part of sand —Anton Van Leeuwenhoek, 1684 300 years ago, Anton Van Leeuwenhoek developed the first prototype of today’s microscope and observed for the first time, life invisible to the naked eye Now we know that these organisms are all around us in teeming millions, and it is their presence (if you call a company of a million or more cells- presence!) that keeps the food chain alive They are our evolutionary ancestors, and we believe that learning about these unicellular creatures would help answer so many of our questions about life After all, they have been here for a couple of billion years! Studying them has helped us lay down some rules that govern life and survival Laws of nature, we’ve learnt, allow only conditional proliferation of organisms All forms of life require certain conditions, both internal and external, to live and proliferate Food, space and environment play an important role in survival Even oxygen, a life nurturing chemical could be toxic for survival While anaerobes cease to thrive in the presence of oxygen, aerobes can tolerate oxygen only up to particular threshold concentrations Aerobes, however, employ antioxidants that can nullify oxygen species that are formed in high oxygen concentrations (Imlay, 2003) Understanding this phenomenon of en1 vironment induced cell death, has been a fascinating study for evolutionary biologists and microbiologists due to the ease of experimenting with microorganisms and for the wealth of evolutionary history we uncover by studying them Some of these micro-organisms, however, -inadvertently as a part of their life cycle- are harmful to us They can cause serious illnesses that shut down our immune defense mechanisms, eventually leading us to death We have used our knowledge about them and turned it against them By means of antibiotics, we are able to create inhospitable conditions for their survival and ‘induce microbial death’ Scientists have, since its miraculous discovery, probed into mechanisms of antibiotic action The idea of this is to manufacture more of these substances and ward off threats to human health Antibiotic induced cell death has been an area of commercial, intellectual, and scientific interest Studying cell death mechanisms has noticed accentuated interest, especially in the face of the emerging antibiotic resistance Bacteria are constantly reinventing themselves to escape antibiotic mediated damage/death The existing antibiotic weaponry is failing us, and we are once again thrust into a war against these bugs Before the pre-antibiotic world becomes today’s reality, we must find ways to combat them Thus, reverting back to studying basic bacterial biology and understanding mechanisms of cell survival and cell death seems more fitting now, than ever tive stress has been shown in organisms such as Saccharomyces cerevisiae (Davidson, Whyte, Bissinge & Schiestl, 1996), Aspergillus niger (Abrashev, Pashova, Stefanova, Vassilev, Dolashka-Angelova & Angelova, 2008) and even E coli (Privalle & Fridovich, 1987) Further research needs to be conducted to explore the dynamics of these processes in Mycobacteria and other organisms There is also a possibility that M smegmatis might initiate programmed cell death upon exposure to stress (antibiotic) such as the MazEF pathway characterised in E coli The MazEF system is a toxin-antitoxin system encoded in the chromosome where in MazE is a labile anti-toxin while MazF is a stable toxin Under normal conditions, MazE inhibits the action of MazF Under stress, the toxin MazF is induced and large populations of cells die in a ROS dependent or independent manner This is believed to be an altruistic mechanism of cell survival via quorum sensing as cells are sacrificed in order to allow a small population of cells to thrive when favorable conditions return However, E coli also outlines a-selfish mode of survival whereupon individual cells repair their DNA to try and outlive stressful conditions This is known as the apoptotic like death (ALD) pathway (Erental, Sharon, & EngelbergKulka, 2012) Complicated as these are, these mechanisms might be employed by bacteria to evade the constant pressure we are imposing through the means of antibiotics and the mycobacterial species might well employ such convoluted mechanisms to survive Thus, additional studies looking into such pathways might just be worthwhile to pursue 54 The impetus for exploring such cell death pathways is to understand bacterial responses to antibiotic induced stress Research teams over the world have taken up the different approaches to comprehend cell survival techniques and cell death pathways to make antibiotics that could outlast bacterial recovery mechanisms While some teams such as ours seek out to a step by step analysis of bacterial responses, other teams have alternatively tried to understand quorum sensing and finding ways to silence and tame bacteria such that they cease to be pathogenic It seems to be worthwhile to invest time, energy and manpower in such studies that could generate a knowledge resource which could be in turn used to design better drugs and giving better treatment to diseasedindividuals 55 Conclusion The threat of antibiotic resistance has provided the right stimulus to explore bacteria ROS induced cell death has been a fascinating area for scientists and developing antibiotics specific to bacterial targets that could trigger reactive oxygen intermediates is an enticing idea Thus, the study by Kohanski et al (2007) was groundbreaking and revolutionary in its own right and evaluating this pathway in other bacteria is an important area of research In the present study, we have developed an assay to detect the deleterious hydroxyl radicals produced in Mycobacterium smegmatis Our study indicates that the applied cidal concentrations of fluoroquinolones trigger a common cell death pathway that produces significant amounts of hydroxyl radicals that causes bacterial cell death However, hydroxyl radical induced death can be prevented, in vivo, by the application of fenton reaction inhibitors The fenton inhibitors, thiourea and dypridyl prevent the formation of hydroxyl radicals upon ciprofloxacin treatment thereby increasing cell survival On the other hand, contrary to the Kohanski study, cidal concentrations of drugs kanamycin, isoniazid and ethambutol did not induce hydroxyl radicals This suggests that in contrast to E coli and S aureus, not all cidal drugs kill via the generation of hydroxyl radicals Mycobacteria, in contrast to Gram positive and Gram negative bacteria appear to 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higher fluorescence intensity thereby indicating the production of hydroxyl radicals as detected by the probe The samples were collected using BD FACS Calibur flow cytometer with a 488 nm argon laser and a 515–545 nm emission filter(FL1) at low flow rate Atleast 50,000 events were collected for each sample PMT voltage settings used: E00 (FSC), 360 (SSC), 825 (FL1) 67 Figure 16: The experiment was conducted to replicate the findings by Kohanski (2007) Log phase cultures of E coli were treated with 5µg/mL kanamycin and assessed for hydroxyl radicals at hours post drug application The orange curve indicated by the black arrow head on the histogram shows a shift to higher fluorescence intensity thereby indicating the production of hydroxyl radicals as detected by the probe The samples were collected using BD FACS Calibur flow cytometer with a 488 nm argon laser and a 515–545 nm emission filter(FL1) at low flow rate Atleast 50,000 events were collected for each sample PMT voltage settings used: E00 (FSC), 360 (SSC), 825 (FL1) 68 ... TABLE OF CONTENTS ABSTRACT v LIST OF TABLES vi LIST OF FIGURES vii INTRODUCTION 1.1 CELLULAR ENVIRONMENT INDUCED DEATH 1.2 ANTIBIOTIC INDUCED CELL DEATH 1.3 THE COMMON DEATH PATHWAY- GOING AGAINST... a in e d IN H h IN H h 5 1 0 5 0 0 * IC M IC IN IN H IN H IN H H /2 M M IC IC M IC l /5 IN H IN C H o n M tr o IC M IC IN IN H H M M IC IC M /2 IN IN H H C o /5 n M tr o IC l * vi iii IN H h IN. .. Antibiotic induced cell death has been an area of commercial, intellectual, and scientific interest Studying cell death mechanisms has noticed accentuated interest, especially in the face of the emerging