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Antimicrobial Peptide Daptomycin and Its Inhibition by Pulmonary Surfactant: Biophysical Studies Using Model Membrane Systems

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Antimicrobial Peptide Daptomycin and Its Inhibition by Pulmonary Surfactant: Biophysical Studies Using Model Membrane Systems Antimicrobial Peptide Daptomycin and its Inhibition by Pulmonary Surfactant: Biophysical Studies using Model Membrane Systems by Brenda Yasie Lee A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Biology (Nanotechnology) Waterloo, Ontario, Canada, 2017 © Brenda Yasie Lee 2017 EXAMINING COMMITTEE MEMBERSHIP The following served on the Examining Committee for this thesis. The decision of the Examining Committee is by majority vote. EXTERNAL EXAMINER: DR. ELMAR JOSEF PRENNER Associate Professor Department of Biological Sciences University of Calgary SUPERVISOR: DR. ZOYA LEONENKO Professor Department of Biology Department of Physics & Astronomy University of Waterloo INTERNAL MEMBER: DR. BARBARA A. MOFFATT Professor Department of Biology University of Waterloo INTERNAL-EXTERNAL MEMBER: DR. MAUD GORBET Associate Professor Department of Systems Design Engineering University of Waterloo MEMBER: DR. MICHAEL PALMER Associate Professor Department of Chemistry University of Waterloo ii AUTHOR’S DECLARATION This thesis consists of material all of which I authored or co-authored: see Statement of Contributions included in the thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. iii STATEMENT OF CONTRIBUTIONS All of the experiments presented in this thesis were conducted by myself and designed by myself with advice and guidance from my committee members: Dr. Zoya Leonenko, Dr. Michael Palmer, and Dr. Barbara Moffatt. All of the figures presented in this thesis were drawn by myself with occasional advice and guidance from Craig Christoff at CryoDragon Inc., unless otherwise noted in the figure caption. Specific exceptions to the above statements are noted as follows: Chapter 5: A portion of the preliminary data for this study was obtained by undergraduate student Victoria Higgins, whom I trained and co-supervised. However, all of the experiments were redone using new stock solutions and updated experimental protocols. For these new experiments, undergraduate students Jeff Lam and Maureen Li (whom I trained and co- supervised) helped with a portion of sample preparation and the acquisition of preliminary fluorescence emission spectra. Chapter 6: The monolayer compression isotherms and insertion assays obtained for this study were primarily performed and repeated by myself, with support from Jeff Lam and Maureen Li for data collection of a subset of experiments pertaining to lung surfactant and bacterial membrane lipid model systems. The antibiotics daptomycin and CB-182,462 used for this work were generously donated by Dr. Jared Silverman of Cubist Pharmaceuticals (now a subsidiary of Merck & Co.). iii ABSTRACT Daptomycin is a lipopeptide antibiotic that is clinically used to treat severe infections caused by Gram-positive bacteria. It is highly potent against resistant strains of bacteria such as methicillin- resistant Staphylococcus aureus. However, in cases of community-acquired pneumonia (a leading cause of death worldwide), daptomycin is somehow inhibited by lung surfactant and therefore unable to exert its bactericidal activity against Streptococcus pneumoniae, the primary cause of this disease. This thesis presents the successful development of lipid model systems to mimic the lipid composition of S. pneumoniae bacterial membranes, human cell membranes, and both synthetic and natural lung surfactant. Experiments were performed that help to elucidate the basis for daptomycin’s inhibition by lung surfactant, culminating in a new, detailed model of daptomycin sequestration that summarizes the findings from these studies. Daptomycin is believed to be sequestered by lung surfactant and has been shown to insert into this surfactant. Fluorescence spectroscopy experiments were used to test the interaction of daptomycin with different lipid model membranes in the presence of calcium. It was discovered that at physiologically relevant calcium concentrations, daptomycin induced larger changes in fluorescence intensity with lung surfactant than in the bacterial membrane and human membrane models. This suggests that daptomycin has a greater affinity for lung surfactant at lower calcium concentrations, which may account for its inhibition in these conditions. Using Langmuir-Blodgett monolayer techniques, studies were performed on how daptomycin affects monolayer properties. Compression isotherms provided data on monolayer compressibility, and it was found that daptomycin and calcium reduce the compressibility of lung surfactant monolayers, possibly improving its function. Constant-area insertion assays provided additional data that verified daptomycin’s avid binding to lung surfactant at low calcium concentrations. iv Scanning probe microscopy techniques were employed to obtain atomic force microscopy and Kelvin probe force microscopy images for monolayers in air. In the presence of daptomycin and calcium, the lung surfactant monolayers exhibited multilayer formation and increased electrical surface potential. Atomic force microscopy images taken of model lipid bilayers in liquid show multi-bilayer formation for the lung surfactant bilayers in the presence of daptomycin and calcium. This provides further evidence that daptomycin and calcium induce multilayer formation in lung surfactant. These findings allowed for the development of a novel model of daptomycin inhibition by lung surfactant. In the presence of physiological levels of calcium, daptomycin binds to lung surfactant and is sequestered. This binding causes a decrease in lung surfactant compressibility, allowing it to easily form multilayers that effectively reinforce the sequestration of daptomycin. The lipid models, methods, and experimental protocols developed in this thesis will help foster future studies in the field of membrane biophysics. v ACKNOWLEDGEMENTS This doctoral dissertation represents a culmination of over six years’ worth of experiences and milestones at the University of Waterloo. First and foremost, I would like to thank my supervisor, Dr. Zoya Leonenko, for taking me under her wing. Without her constant support and guidance, this thesis would not have been possible. I would also like to thank Dr. Leonenko for her commitment to her lab members and providing each and every one of them, including myself, with as many opportunities as possible to help them progress further in their future careers by encouraging scientific investigation and making sure we had all the necessary skills and knowledge to do so. The freedom she has provided us with has allowed me to pursue multiple passions of mine: teaching and scientific graphic design, all of which has helped me grow as a researcher and educator. As a recipient of her continuous mentorship and leadership, I want to express my sincerest gratitude to Dr. Leonenko for playing such a large role in shaping my future. I can never thank her enough for all that she has done for me. Thank you to Dr. Michael Palmer for opening the doors to his lab so that I could benefit from the expertise of his team in fluorescence spectroscopy, daptomycin, and its derivatives. I am also grateful to Dr. Palmer for his collaborative support and the many helpful discussions we have had regarding experimental procedures and results. I am thankful for the opportunity to have worked in his lab and to have experienced his mentorship and sense of humour firsthand. I would also like to thank Dr. Barbara Moffatt for her ongoing support and mentorship throughout my journey as both a graduate student and community helper. Dr. Moffatt provided me with invaluable support and critique related to experimental design and data analysis, all of which helped me fine-tune my experiments and ensure the implementation of strict protocols and data collection procedures for my project. vi In our lab, I would first like to thank Dr. Ravi Gaikwad, who introduced me to the JPK atomic force microscope when I first joined Dr. Zoya Loenenko’s lab. Ravi spent months training me on the JPK and helped a nervous undergraduate student gain her ground by providing constant moral support and friendship along the way. I am grateful for all of his mentorship in helping me gain expertise in atomic force microscopy and appreciating scientific investigation. I would also like to than Drs. Elizabeth Drolle and Robert D.E. Henderson for helping to train me not only on the AIST-NT atomic force microscope, but also the Langmuir-Blodgett trough. I appreciate all of the helpful discussions and friendly comradery we had along the way. Next, I would like to offer my deepest gratitude to Jeff Lam, Maureen Li, and Victoria Higgins for their collaborative support and assistance with my project. Your friendship and support have been invaluable throughout these years. Thank you to all of my labmates (Morgan, Stephen, Francis, Jennifer, Jingsi, Nancy Mei, Kelsey, Vince, Ravi, Simon, Nancy Elewa, and Tavo, just to name a few) for making my lab experience one that I will cherish forever. I have also worked with numerous researchers outside of our lab. Thank you to Dr. Jared Silverman for not only supplying daptomycin and CB-182,462 for my project, but for the helpful discussions we had regarding these peptides. I have greatly benefited from Dr. Silverman’s expertise. Thank you to Dr. Sid Ragona and Dr. Marc Richter for providing additional training and support for the JPK atomic force
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