Antimicrobial Peptide Daptomycin and its Inhibition by Pulmonary Surfactant

275 Pages·2017·8.16 MB·English

by  Brenda Lee

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
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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.
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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.).
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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.
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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.

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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.
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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 microscope, specifically with regards to imaging in liquid
environments; as well as Dr. Andrey Krayev from AIST-NT for his support and guidance with
the SmartSPM’s AFM and KPFM capabilities. In Dr. Palmer’s lab, I would like to thank Dr.
Oscar TianHua Zhang and Robert Taylor for their training, support and helpful discussions in
all things fluorescence spectroscopy and daptomycin.
On a final note, I would like to thank Craig Christoff for spending countless days, nights, and
weekends in the lab with me while I was running experiments and for providing me with
guidance and support for graphic design and being able to calm my nerves during times of high
stress and looming deadlines. I could not have done this without your moral support.
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DEDICATION

To my amazing father, Richard Lee, for his unwavering support, love and encouragement to
continually strive for academic excellence and to be the best that I can be. Also for his dedicated
effort and selflessness in helping me with Tutoring Beyond Borders while I was immersed in my
studies. You have always been my greatest role model.
To my hardworking mother, Jenny Chiou, for her years of sacrifice and perseverance in making
this family the most unique and loving environment a child could ever wish for. It has been a
tough and formidable journey, we made it through, and it is time to enjoy the fruits of your
labour. You have been an inspiration to all of us.
To my loving mama, Mina Chan, for being my closest friend and confidant since the day I
entered this world. No matter what, no matter where, I could always depend on you because
you were always there for me. It is now time for me to master all of your delicious recipes!
To my sister, Jessie Lee, for showing me that life is precious and that you have to make the most
of it while you still can. I am proud of your bravery in facing the greatest odds, and I can’t wait
to see Ashley and Rosabel grow and reach for the stars.
To my best friend, Craig Christoff, for his constant encouragement and support. You have
shown me the true meaning of how good things come to those who wait. Your dedication to
never giving up and fighting for what you want has encouraged me to do the same, and I look
forward to seeing our business flourish in years to come.
Last but not least, to Dr. Zoya Leonenko, without whom none of this would have been possible.
I am the person I am today because of you and the opportunities you have given me. I am
forever indebted to you for your kindness and support, and I am grateful to call you my lifelong
mentor and friend. We miss you, Marsik.
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TABLE OF CONTENTS

EXAMINING COMMITTEE MEMBERSHIP ............................................................. ii
AUTHOR’S DECLARATION ....................................................................................... iii
STATEMENT OF CONTRIBUTIONS ........................................................................ iii
ABSTRACT ..................................................................................................................... iv
ACKNOWLEDGEMENTS ............................................................................................ vi
DEDICATION ............................................................................................................. viii
TABLE OF CONTENTS ............................................................................................... ix
LIST OF FIGURES ........................................................................................................ xv
LIST OF TABLES ...................................................................................................... xviii
LIST OF ABBREVIATIONS ....................................................................................... xix
LIST OF SYMBOLS .................................................................................................... xxii
1 INTRODUCTION ..................................................................................................... 1
1.1 Antimicrobial Peptides ................................................................................... 3
1.2 Bacterial Resistance ........................................................................................ 6
1.3 Daptomycin ..................................................................................................... 8
1.3.1 History and Development ............................................................................................... 9
1.3.2 Mechanism of Action ..................................................................................................... 12
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