Lysin Based Antimicrobial Peptides Against Acinetobacter Baumannii

95 Pages·2017·12.51 MB·English

by  Mya Thandar

Rockefeller University
Digital Commons @ RU
Student Theses and Dissertations
2017
Lysin Based Antimicrobial Peptides Against
Acinetobacter Baumannii
Mya Thandar
Follow this and additional works at:http://digitalcommons.rockefeller.edu/
student_theses_and_dissertations
Part of theLife Sciences Commons
Recommended Citation
Thandar, Mya, "Lysin Based Antimicrobial Peptides Against Acinetobacter Baumannii" (2017).Student Theses and Dissertations. 390.
http://digitalcommons.rockefeller.edu/student_theses_and_dissertations/390
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 [email protected].
LYSIN BASED ANTIMICROBIAL PEPTIDES AGAINST ACINETOBACTER
BAUMANNII
A Thesis Presented to the Faculty of
The Rockefeller University
in Partial Fulfillment of the Requirements for
the degree of Doctor of Philosophy
by
Mya Thandar
June 2017
© Copyright by Mya Thandar 2017
LYSIN BASED ANTIMICROBIAL PEPTIDES AGAINST ACINETOBACTER
BAUMANNII
Mya Thandar, Ph.D.
The Rockefeller University 2017
Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a
range of nosocomial infections. The recent rise and spread of multidrug resistant A.
baumannii clones has fueled a search for alternative therapies, including bacteriophage
endolysins with potent antibacterial activities. A common feature of these lysins is the
presence of a highly positively charged C-terminal domain with a likely role in
promoting outer membrane penetration. In the current study, we show that the C-terminal
amino acids 108-138 of phage lysin PlyF307, named P307, alone was sufficient to kill A.
baumannii (>3-logs). Furthermore, P307 could be engineered for improved activity, the
most active derivative being P307 (>5-log kill). Both P307 and P307 showed
SQ-8C SQ-8C
high in vitro activity against A. baumannii in biofilms. Moreover, P307 exhibited
SQ-8C
MICs comparable to levofloxacin and ceftazidime and acted synergistically with
polymyxin B. While the peptides were shown to kill by disrupting the bacterial
cytoplasmic membrane, they did not lyse human red blood cells or B cells; however,
serum was found to be inhibitory to lytic activity. In a murine model of A. baumannii
skin infection, P307 reduced the bacterial burden by ~2-logs in 2 h. This study
SQ-8C
demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat
topical infections and remove biofilms caused by Gram-negative pathogens.
To my grandparents
To my parents
To Neko
iii
ACKNOWLEDGMENTS
Foremost, I would like to thank Dr. Vincent Fischetti for being the most
understanding and supportive mentor. Words could not express my gratitude to him. He
has always been available for discussing my work and each discussion has challenged
and cultivated me to become a better scientific thinker. Throughout my graduate studies,
his wisdom has guided me and his encouragement has motivated me. It has been a
privilege having him as my advisor and mentor.
I am grateful to my faculty advisory committee: Dr. Luciano Marraffini and Dr.
Howard Hang. Their insights and counsels have been stimulating and invaluable for my
progress. I would also like to thank Dr. Ryland Young for kindly accepting a position on
my committee as the external examiner.
I am thankful to Dr. Rolf Lood and Benjamin Winer for showing me the ropes in
the lab. They have played crucial roles in helping me launch my thesis project. Dr. Chad
Euler has patiently guided and assisted me in conducting in vivo experiments. I am also
grateful to Clara Eastby and my summer students Ravenne Reid and Gabriella Balaa for
their excellent assistance. I am much obliged to the past and present members of the
Fischetti Laboratory who have contributed to my project by providing valuable feedbacks
and ideas. Thanks to the cordial and collaborative space they provided, this journey has
been an enjoyable one. I also want to thank Dr. Michael Wittekind and Dr. Raymond
Schuch for their useful discussions and suggestions and ContraFect Corporation for the
funding support.
I am incredibly grateful to the Rockefeller community for providing the ideal
place to hone my scientific skills. The Proteomics Resource Center, the Electron
iv
Microscopy Resource Center, the Comparative Bioscience Center and the Hospital have
contributed their technical expertise to my research. The Housing and Security
Departments and the mailroom have ensured my safe and comfortable living
environment. Everyone in the Dean’s Office – Dr. Sidney Strickland, Dr. Emily Harms,
Dr. Andrea Morris, Marta Delgado, Kristen Cullen, Cris Rosario and Stephanie
Fernandez – have enabled me to fulfill my academic aspirations without an issue.
I would like to thank my past mentors Dr. Seth Darst, Dr. Joseph Osmundson, Dr.
Lilian Hsu and Tenaya Vallery who have shared with me the joy of scientific discovery.
Lastly, I would like to thank all my friends and family who have sustained me with their
unwavering love and kindness.
v
TABLE OF CONTENTS

DEDICATION .................................................................................................................. iii
ACKNOWLEDGMENTS ............................................................................................... iv
TABLE OF CONTENTS ................................................................................................ vi
LIST OF FIGURES .......................................................................................................... x
LIST OF TABLES .......................................................................................................... xii
1 CHAPTER 1 – INTRODUCTION ............................................................................ 1
1.1 Acinetobacter baumannii ...................................................................................... 1
1.1.1 History ............................................................................................................ 1
1.1.2 Prevalence and pathogenesis ........................................................................... 4
1.1.3 Virulence and antibiotic resistance .................................................................. 6
1.1.4 Treatment ......................................................................................................... 7
1.2 Alternative antibacterial agents ............................................................................. 9
1.2.1 Bacteriophages ................................................................................................ 9
1.2.2 Phage lysins ................................................................................................... 12
1.2.3 Antimicrobial peptides .................................................................................. 16
1.3 AIMS ................................................................................................................... 17
2 CHAPTER 2 – LYSIN-BASED ANTIMICROBIAL PEPTIDES ....................... 18
2.1 MATERIALS AND METHODS ........................................................................ 18
2.1.1 Peptides ......................................................................................................... 18
2.1.1.1 Identification, modifications and structural predictions .......................... 18
2.1.1.2 Synthesis .................................................................................................. 20
vi
2.1.2 Activity ......................................................................................................... 21
2.1.2.1 Bacterial strains and growth conditions .................................................. 21
2.1.2.2 Bactericidal assays ................................................................................... 22
2.1.2.3 MIC, resistance and synergy assays ........................................................ 23
2.2 RESULTS ............................................................................................................ 25
2.2.1 Structural analyses of peptides ...................................................................... 25
2.2.2 Activity ......................................................................................................... 26
2.2.2.1 Bactericidal assays ................................................................................... 26
2.2.2.2 MIC, resistance and synergy assays ........................................................ 33
2.3 ACKNOWLEDGMENTS .................................................................................. 34
3 CHAPTER 3 – INFLUENCING FACTORS ON BACTERICIDAL
ACTIVITIES ................................................................................................................... 35
3.1 MATERIALS AND METHODS ........................................................................ 35
3.1.1 Inter-peptide disulfide bond formation .......................................................... 35
3.1.2 pH-dependent sensitivity of E. coli and K. pneumoniae ............................... 35
3.1.3 DNA-binding assay ....................................................................................... 35
3.1.4 Transmission electron microscopy (TEM) .................................................... 36
3.1.5 SYTOX green uptake assay .......................................................................... 36
3.2 RESULTS ........................................................................................................... 37
3.2.1 Inter-peptide disulfide bond formation .......................................................... 37
3.2.2 pH-dependent sensitivity of E. coli and K. pneumoniae ............................... 37
3.2.3 DNA-binding assay ....................................................................................... 40
3.2.4 Transmission electron microscopy (TEM) .................................................... 41
vii
3.2.5 SYTOX green uptake assay .......................................................................... 41
3.3 ACKNOWLEDGMENTS ................................................................................... 41
4 CHAPTER 4 – SAFETY AND IN VIVO ACTIVITY ........................................... 44
4.1 MATERIALS AND METHODS ........................................................................ 44
4.1.1 Cytotoxicity assays ........................................................................................ 44
4.1.2 Endotoxin assay ............................................................................................. 45
4.1.3 Bactericidal activity in plasma and its components ...................................... 45
4.1.4 Mouse skin infection ..................................................................................... 46
4.2 RESULTS ............................................................................................................ 47
4.2.1 Cytotoxicity assays ........................................................................................ 47
4.2.2 Endotoxin assay ............................................................................................. 47
4.2.3 Bactericidal activity in plasma and its components ...................................... 50
4.2.4 Mouse skin infection ..................................................................................... 50
4.3 ACKNOWLEDGMENTS ................................................................................... 54
5 CHAPTER 5 – DISCUSSION ................................................................................. 55
5.1 Lysin-based antimicrobial peptides ..................................................................... 55
5.1.1 Sequence and structure .................................................................................. 55
5.1.2 In vitro activity .............................................................................................. 56
5.2 Mechanism of action ............................................................................................ 57
5.3 In vivo activity ..................................................................................................... 59
5.4 Opportunities for improvement ........................................................................... 59
5.5 Potential practical uses ........................................................................................ 61

viii