Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2012 Roles of the Methylcitrate and Methylmalonyl- COA Pathways in Mycobacterial Metabolism and Pathogenesis Manisha Ulhas Lotlikar Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Lotlikar, Manisha Ulhas, "Roles of the Methylcitrate and Methylmalonyl-COA Pathways in Mycobacterial Metabolism and Pathogenesis" (2012). Student Theses and Dissertations. Paper 245. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. ROLES OF THE METHYLCITRATE AND METHYLMALONYL-COA PATHWAYS IN MYCOBACTERIAL METABOLISM AND PATHOGENESIS A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Manisha Ulhas Lotlikar June 2012 © Copyright by Manisha Ulhas Lotlikar 2012 ROLES OF THE METHYLCITRATE AND METHYLMALONYL-COA PATHWAYS IN MYCOBACTERIAL METABOLISM AND PATHOGENESIS Manisha Ulhas Lotlikar, Ph.D. The Rockefeller University 2012 Mycobacterium tuberculosis has been a human pathogen for the history of mankind, but we are only now beginning to understand how it is able to survive and persist indefinitely in the host. Understanding carbon metabolism of the pathogen during infection is key, not only as a source of potential drug targets, but also for elucidating the environment in vivo, so that drugs can be tested under relevant conditions. Studies have revealed that, during infection, M. tuberculosis relies on gluconeogenic carbon sources rather than sugars. Fatty acids, cholesterol, and amino acids have all been demonstrated as usable carbon sources in vitro and can all generate propionyl-CoA. The methylcitrate cycle, which, in M. tuberculosis, uses a bifunctional isocitrate lyase/methylisocitrate lyase (ICL/MCL), is one of the two routes for metabolism of propionyl-CoA. A mutant strain of M. tuberculosis lacking the ICL/MCL was rapidly cleared from the lungs of infected mice. However, the upstream enzymes of this pathway have been demonstrated to be dispensable for infection and survival in the mouse model. The methylmalonyl- CoA route of propionyl-CoA utilization can be activated in vitro by addition of the vitamin B12 cofactor of the methylmalonyl-CoA mutase. This route may buffer the loss of the methylcitrate cycle in vivo, depending on B12 availability or production in the host. The work here examines the relative use of the methylcitrate cycle and methylmalonyl-CoA pathways in M. tuberculosis and in the related, non- pathogenic species, M. smegmatis, using genetic mutants of either or both of the metabolic routes. It is shown here that, as for M. tuberculosis, M. smegmatis preferentially uses the methylcitrate cycle for growth on propionate. In the absence of the methylcitrate cycle M. smegmatis, in contrast to M. tuberculosis, can eventually endogenously activate the methylmalonyl-CoA pathway in vitro, presumably through B12 synthesis. In mutants of both species, lacking both pathways, the use of other carbon sources in the media is inhibited in the presence of propionate. This dominant inhibition implies the accumulation of toxic metabolites derived from the inability to metabolize propionate, as has been suggested by previous studies. To detect propionate-derived intermediates, metabolite analysis by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in this study. Accumulation of these metabolites under propionate exposure was identified in a strain of M. smegmatis impaired in both metabolic routes, but not in the wild-type. These studies also revealed similar accumulation under glucose growth, where the mutant strain displayed a slight growth defect, and also under no-carbon conditions, where the mutant demonstrated a survival defect compared to wild-type. These findings suggest a role of the propionate pathways for endogenously derived propionyl-CoA as well as during starvation- induced amino acid and/or fatty acid mobilization. The M. tuberculosis mutant strains generated here were tested in the mouse infection model. The methylmalonyl-CoA mutase was found to be individually dispensable for growth in vivo. However, a strain with the additional deletion of the methylcitrate cycle was attenuated during the early stage of infection and caused less tissue pathology, even after the bacterial burden reached wild-type levels. While propionate metabolism may not be required per se for in vivo growth, the suggested accumulation of toxic intermediates, demonstrated here in M. smegmatis, may indicate a required role for ICL/MCL in M. tuberculosis for detoxification of propionyl-CoA in vivo. ACKNOWLEDGEMENTS In a sentence, Prof. John McKinney convinced me that I could benefit humanity through tuberculosis research. In the years since, there have been many subsequent sentences - to the effect of superb scientific advising, absorbing discussions, and consistent mentorship. I am grateful to have had the opportunity to learn from him and work in his lab, which he actively orients towards exciting and quality science. I appreciate his patience in this project, which, like M. tuberculosis, grew with a slow but steady doubling time, and for his flexibility with letting me work the ‘night shift’. In addition to John’s scientific views, I have greatly admired his integrity and his real pursuit of productive collaborations. This has been to the great benefit of my work. I have had the privilege to work in the lab of Prof. Uwe Sauer at the ETH Zurich, whose expertise led to some of the most exciting findings in my research. I would like to specifically acknowledge Dr. Jörg Büscher and Michael Zimmermann for their invaluable collaboration on this work. A mutual interest in propionate metabolism also led to our fruitful collaboration with the laboratory of Prof. Valerie Mizrahi at the University of Cape Town, including Dr. Suzana Savvi, Dr. Digby Warner, Dr. Krishnamoorthy Gopinath, and Atica Moosa. I have been fortunate to work in the presence of those willing to share their scientific talents, insight, and experience. Dr. Anna Upton and Dr. Anna Tischler have been mentors and role models, in everything from BSL3-training to scientific iii reasoning. In my every day work, I profit from being just a bay-away from many keen and kind scientific minds, including Cyntia DePiano, Meltem Elitas, and Neeraj Dhar, and those in my Metabolic subgroup: Emre Ozdemir, Tarun Chopra, Paul Murima, and Zeljka Maglica. I am indebted to Prof. Dianne Newman at Caltech for having introduced me to the fascinating world of environmental microbiology through her dedication to teaching. The opportunity she gave me to work in her lab as an undergrad is certainly what revived my love of science at a critical time, and her mentorship inspired me to continue onwards to grad school. The challenges of working with anaerobic bacteria helped prepare me for the difficulties of M. tuberculosis, not just in too-large glovebox-gloves presaging too-large Tyvek suits. In my extracurricular studies of carbon metabolism, I was joined by Dr. Jeffrey Chen and Jocelyne Lew, head curator of Tuberculist, in extending rigorous scientific curiosity to the nutrient availability in our Lausannois environment. In studies of geography and earth science, I have been accompanied by Dr. Sachin Kotak and Sveta Chakrabarti, scientists and badminton players, both. Through the quarter of my lifetime spent on this work, I am immensely grateful for the friends who have known me for over half my life, for the constancy of their support and reliability of their hilarity, that make up for the inconstancies and minor tragedies of experimental science; Sasha Sadikot, Nicole Pusateri, Chi Hae Park, Cathy Serpico, and Eda Uca. iv Every new year that passed in my PhD has fulfilled the promise of a new experience in a new country with the same collegial crew cultivated from our college days; Matt Johnston, David McKinney, Jared Gabor, Peter Samuelson, and Kevin Duncklee. I am especially grateful for the friendship of Bernadette Heyburn, whom, by cosmic CERN-centric coincidence, was expatriating in Switzerland at the same time as myself. For the seamless transition to a trans-Atlantic PhD I would like to thank the Rockefeller University Dean’s Office (Kristen, Cris, Marta, Emily, Sid, Michelle, and Amber) as well as Suzanne Lamy at EPFL. I would like to thank Prof. George Cross and Prof. Howard Hang for serving on my committee, providing their guidance and insight every year, and to Prof. Sabine Ehrt for the honor of her expertise as my external examiner. Instrumental in helping me to get to this point: To my parents, who always have and always would support me in anything, whether I am a country or an ocean apart. To Alpa, to whom I attribute my love of learning, who brought home to share every piece of information she learned at school. To Nimish, Piyusha, Madhavi mami, and Sandeep mama, with whom I have enjoyed the extended support of an extended family. And to Matt, for the brilliance, the humor, the support, of the every single day type, especially for the many days that turn to many years. Thank you all. v TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................