novel synthetic antimicrobial peptides with in-vivo and in-vitro activity against streptococcus ...

301 Pages·2013·10.74 MB·English

NOVEL SYNTHETIC ANTIMICROBIAL PEPTIDES WITH
IN-VIVO AND IN-VITRO ACTIVITY AGAINST
STREPTOCOCCUS PNEUMONIAE

LE CHENG FOH

THESIS SUBMITTED IN FULFILMENT
OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

FACULTY OF MEDICINE
UNIVERSITY OF MALAYA
KUALA LUMPUR

2013

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UNIVERSITI MALAYA
ORIGINAL LITERARY WORK DECLARATION

Name of Candidate: LE CHENG FOH (I. C. No: 851213-14-6157)
Registration/Matric No: MHA100037
Name of Degree: DOCTOR OF PHILOSOPHY (Ph.D)
Title of Project Paper/Research Report/Dissertation/Thesis: NOVEL SYNTHETIC
ANTIMICROBIAL PEPTIDES WITH IN-VIVO AND IN-VITRO ACTIVITY
AGAINST STREPTOCOCCUS PNEUMONIAE
Field of Study: MEDICAL MICROBIOLOGY

I do solemnly and sincerely declare that:

(1) I am the sole author/writer of this Work;
(2) This Work is original;
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for permitted purposes and any excerpt or extract from, or reference to or reproduction
of any copyright work has been disclosed expressly and sufficiently and the title of the
Work and its authorship have been acknowledged in this Work;
(4) I do not have any actual knowledge nor do I ought reasonably to know that the
making of this work constitutes an infringement of any copyright work;
(5) I hereby assign all and every rights in the copyright to this Work to the University of
Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that
any reproduction or use in any form or by any means whatsoever is prohibited without
the written consent of UM having been first had and obtained;
(6) I am fully aware that if in the course of making this Work I have infringed any
copyright whether intentionally or otherwise, I may be subject to legal action or any
other action as may be determined by UM.

Candidate„s Signature: ___________________ Date: ___________________

Subscribed and solemnly declared before,

Witness„s Signature: _____________________ Date: ___________________

Name:

Designation:

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ABSTRACT
Streptococcus pneumoniae is a major human bacterial pathogen causing life
threatening diseases such as meningitis, bacteremia, and pneumonia. Younger children
are heavily affected by pneumococcal infections. Pneumococcal conjugate vaccines
(PCVs) are serotype-specific and require data on local serotype distribution to predict
the coverage of PCVs. Moreover, the escalating incidence of antibiotic resistance S.
pneumoniae has prompted the development of novel antimicrobial agents.
Antimicrobial peptides (AMPs) have been increasingly recognized as the new
generation antibiotics due to their potent antimicrobial activity. In this study, we sought
to design novel synthetic antimicrobial peptides against S. pneumoniae and determine
the in vitro and in vivo activities of the peptides.
A collection of 151 pneumococcal clinical isolates from University of Malaya
Medical Centre, Kuala Lumpur were serotyped using multiplex PCR and the penicillin
susceptibility was determined using agar dilution method. Biocomputational tools were
employed to design the peptides based on the publicly available AMPs. The designed
AMPs were tested for minimum inhibitory concentration, bacterial killing kinetics, and
synergism activity. Broad spectrum antibacterial activity against eight common bacterial
pathogens was also determined. Morphological changes of pneumococcal cells were
observed using transmission electron microscopy. The designed AMPs were also
assessed for hemolytic and cell cytotoxicity. In vivo therapeutic efficacy and acute
toxicity of the peptides were assessed using an in-house mouse model.
Serotypes 19F and 23F were the two prevailing serotypes among Malaysian
population with serotype 19F significantly associated with penicillin resistance.
Penicillin-nonsusceptible S. pneumoniae constituted half (50.3%) of the total isolates.
and as high as 87.5% of penicillin-resistant S. pneumoniae (PRSP) were PCV7-vaccine
serotypes. From the peptide designs, antimicrobial testing showed that the five hybrid
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peptides (DM1-5) exhibited strong antipneumococcal activity irrespective of penicillin
susceptibility of the isolates. These isolates included the highly prevalent serotype 19F.
The pneumococcal killing rates were higher than penicillin by 38 – 64%, 54 – 76%, and
30 – 57% for penicillin-resistant, intermediate, and susceptible isolates at 30 min
posttreatment, respectively. The DMs and other peptides produced synergism in
combination with penicillin against S. pneumoniae and displayed broad spectrum
antibacterial activities. The DMs induced overwhelming cellular damages leading to
cell death. Besides, DMs exhibited low hemolytic activity (HC >250 μg/ml) with
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varying degrees of cell cytotoxicity against NL20 and A549 cell lines. Notably, DM3
(40 mg/kg) given via intraperitoneal route at 12 hrs interval for three dosing regimens
protected 50% of mice from lethal systemic infection by a PRSP strain. Interestingly,
combination therapy using low doses (10mg/kg and 20mg/kg) DM3 and penicillin
showed therapeutic synergism with 20-50% higher survival rates than the sum of the
standalone treatments. Complete protection (100%) was achieved with DM3 (20 mg/kg)
– penicillin (20 mg/kg) combination.
Pneumococcal infections continue to affect humans and antibiotic-resistant S.
pneumoniae would further complicate the treatment outcomes. The current study
demonstrated that AMPs represent the promising new generation antibiotics as
standalone therapeutics or in combination with conventional antibiotics. Novel designed
AMPs candidates can be designed following careful designing approaches to generate
peptides with high therapeutic potential to be further developed into clinically useful
antibiotics.

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ABSTRAK
Streptococcus pneumoniae merupakan sejenis patogen bakteria yang boleh
menyebabkan pelbagai jenis penyakit yang mengancam nyawa manusia seperti
meningitis, bakteremia, dan pneumonia. Kanak-kanak merupakan golongan utama yang
sering diancam oleh jangkitan pneumokokal. Keberkesanan “pneumococcal-conjugate
vaccine (PCV)” adalah spesifik kepada jenis serotype dan data mengenai taburan
serotype tempatan amat diperlukan supaya liputan PCVs dapat dianggarkan. Selain itu,
isu-isu peningkatan rintangan antibiotik S. pneumoniae telah meningkatkan permintaan
terhadap antibiotik baru. Peptide antimikrobial (AMPs) mempunyai aktiviti antimikrob
yang tinggi dan telah kian diiktiraf sebagai antibiotik generasi baru. Dalam kajian ini,
kami telah mereka peptida sintetik terhadap S. pneumoniae dan menentukan aktiviti-
aktiviti in vitro dan in vivo peptida tersebut.
Sebanyak 151 isolat pneumokokal klinikal dari Pusat Perubatan Universiti
Malaya, Kuala Lumpur telah diserotypekan menggunakan PCR multipleks dan tahap
rintangan penisilin ditentukan dengan kaedah pencairan agar. Teknik biokomputer
digunakan untuk mereka peptida baru berdasarkan peptida yang sedia ada. Seterusnya,
peptida sintetik tersebut diuji untuk menentukan “minimum inhibitory concentration
(MIC)”, kinetik penghapusan bakteria, dan aktiviti sinergi. Peptida sintetik tersebut juga
diuji untuk keberkesanan antibakteria specktrum luas terhadap lapan bakteria patogen.
Perubahan morfologi sel pneumokokal telah divisualisasikan dengan menggunakan
mikroskop elektron transmisi (TEM). Selain itu, tahap hemolitik dan ketoksikan peptida
sintetik tersebut terhadap sel-sel manusia turut ditentukan. Keberkesanan terapeutik dan
ketoksikan in vivo telah dinilai menggunakan model tetikus.
Serotype 19F dan 23F merupakan dua jenis serotype lazim di kalangan penduduk
Malaysia. Serotype 19F adalah dikaitkan dengan rintangan penisilin. Separuh daripada
jumlah isolat merupakan S. pneumoniae yang toleran kepada penisilin (50.3%).
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Sebanyak 87.5% S. pneumoniae yang rintang penisilin (PRSP) merupakan serotype
yang terkandung dalam vaksin PCV7. Ujian antimikrobial menunjukkan bahawa aktiviti
DAMP7 adalah sederhana manakala lima peptida hibrid (DM1-5) mempamerkan
aktiviti antipneumococcal yang kuat terhadap semua isolat pneumokokal tanpa
bergantung kepada rintangan penisilin isolat termasuk serotype 19F. Kadar
penghapusan pneumokokal adalah lebih tinggi daripada penisilin dan mencapai 38 -
64%, 54 - 76%, dan 30 - 57% bagi isolat resistan, sederhana, dan sensitif penisilin
masing-masing selepas dirawat untuk 30 min. DAMP7 dan DMs dapat menghasilkan
kesan sinergi terhadap S. pneumoniae semasa dikombinasikan bersama penisilin dan
juga mempunyai aktiviti antibakteria spektrum luas. DMs juga menyebabkan kerosakan
dinding dan membran sel yang akhirnya membawa kepada kematian sel. Peptida hibrid
tersebut menunjukkan aktiviti hemolitik yang rendah (HC > 250 μg/ml) dan tahap
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ketoksikan yang berlainan terhadap sel-sel NL20 dan A549. Rawatan DM3 (40 mg/kg)
secara intraperitoneal pada setiap selangan 12 jam untuk tiga regim dapat
menyembuhkan 50% tetikus daripada jangkitan maut sistemik oleh PRSP. Terapi
combinasi dengan dos rendah DM3 dan penicillin menunjukkan sinergi teraputik di
mana kadar pelindungan sebanyak 20 – 50% lebih tinggi daripada rawatan tunggal
dicatatkan. Perlindungan menyeluruh (100%) dapat dicapai dengan kombinasi DM3 (20
mg/kg) – penicillin (20 mg/kg).
Jangkitan pneumokokal akan terus mengancam manusia dan dijangka akan
merumitkan lagi rawatan untuk penyakit-penyakit yang melibatkan S. pneumoniae
rintang antibiotik. Kajian ini menunjukkan bahawa AMPs merupakan calon antibiotik
generasi baru yang berpotensi untuk rawatan secara tunggal ataupun diformulasikan
dengan antibiotik konvensional. Penyelidikan secara systematik dan teliti dapat
membantu menghasilkan AMPs yang mempunyai potensi terapeutik yang tinggi untuk
kegunaan klinikal.
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ACKNOWLEDGEMENT
First and foremost, I would like to express my deepest appreciations to both of my
supervisors, Prof. Dr. Shamala Devi and Associate Prof. Dr. Mohd Yasim Md Yusof,
Department of Medical Microbiology, University of Malaya. They have provided me
with guidance, encouragement, and constant supports throughout the course of my
research study. They have enlightened me and drive me to another peak of my life
which makes me a stronger and better person that I was before this.
Towards the completion of this study, I have been fortunate enough to have a lot
of lovely and generous lab mates and friends in my lab who always be there to offer
their helps especially Dr. Wang Seok Mui, Dr. Deepa Anbazhagan, Dr. Mohammed, Dr.
Ramapraba, Ms. Lee Sau Har, Mr. Tang Yin Quan, Mr. Tan Wee Chee, Ms. Thamil
Vaani, Ms. Adeline Yeo, Ms. Anusyah and others. In addition, fellow friends from my
department and the faculty have provided me with various aspects of sincere helps. I
would like to thank Ms. Vanitha Mariappan, Mr. Choh Leang Chung, Mrs. Kumutha,
and Mr. Ong Guang Han for discussing and sharing their ideas and giving assistances
when I am very much in need of. Very special thanks to Mr. Tan Soon Hao from the
Department of Pathology, Mrs. Lee Mui Li and Ms. Yeannie Yap Hui Yeng from the
Department of Molecular Medicine, and Mr. Vincent Chong Vui King from the Faculty
of Dentistry for their supports in research works and in daily life matters.
This thesis would not have been possible without Prof. Dr. Mahmood Ameen
Abdulla Hassan, Department of Biomedical Sciences who have willingly allocated his
busy schedule of daily work to assist in the histopathological examinations of the mice
samples that I have collected for my study. Without his experienced and professional
advices, the interpretation of the histology slides would not have been able to be
completed within the limited time frame before my thesis submission. Special thanks to
Dr. Ong Kien Chai who provided his advices on animal handling issues.
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I also would like to thank the Ministry of Science, Technology and Innovation
(MOSTI), Malaysia research grant (02-01-03-SF0353) and the University of Malaya
Research Grant (RG215-10HTM) and postgraduate research grants (PS187/2009B,
PV077/2011B) which provided the financial supports needed for my research works.
Special thanks to University of Malaya Scholarship Scheme (SBUM) for providing
financial support to complete my study.
Although I couldn‟t name them all, I must mention the friendly staffs and
members from my department, the Electron Microscopy Unit, the animal house, and the
Faculty of Medicine who have directly or indirectly helped out in solving my
documentations or experimental problems. I really appreciate the help by the staffs from
the Veterinary Laboratory Service Unit, Faculty of Veterinary Medicine, Universiti
Putra Malaysia especially Mr. Mohamad Halmi Othman, Mr. Abdullah Misron, and
Puan Jamilah for their kind assistances in processing the mice samples that I sent in
even though I am from a different university.
Finally, I would like to thank my family members and Ms. Yeong Pooi San and
her family members who have been closest to me when my spirit is weak and when my
emotion is down. Thanks for bearing with me, cheering me up, and encouraging me
towards the completion of my study.
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TABLE OF CONTENTS
ABSTRACT ................................................................................................................ iii
ABSTRAK ................................................................................................................. v
ACKNOWLEDGEMENT ............................................................................................ vii
TABLE OF CONTENTS ............................................................................................... ix
LIST OF FIGURES ...................................................................................................... xii
LIST OF TABLES ....................................................................................................... xvi
LIST OF SYMBOLS AND ABBREVIATIONS ..................................................... xviii

CHAPTER 1: INTRODUCTION ..................................................................................... 1
1.1. Overview of Streptococcus pneumoniae ............................................................ 2
1.2. Pneumococcal virulence factors ......................................................................... 3
1.2.1. Pneumolysin ............................................................................................ 3
1.2.2. Choline-binding proteins ......................................................................... 5
1.2.3. Capsular Polysaccharide ......................................................................... 7
1.2.4. Pneumococcal colonization ..................................................................... 9
1.2.5. Pneumococcal infections ....................................................................... 10
1.3. Pneumococcal serotype distribution ................................................................. 14
1.3.1. Pneumococcal vaccinations .................................................................. 14
1.3.2. Pre- and Postvaccination changes in serotype distribution ................... 16
1.3.3. Serotype-specific pneumococcal virulence ........................................... 19
1.4. Antibiotic-resistant Streptococcus pneumoniae ............................................... 20
1.4.1. β-lactam resistance ................................................................................ 22
1.4.2. Macrolide resistance.............................................................................. 27
1.4.3. Fluoroquinolones resistance .................................................................. 30
1.5. Antimicrobial peptides ..................................................................................... 33
1.5.1. General properties of antimicrobial peptides ........................................ 33
1.5.2. Structural classes ................................................................................... 36
1.6. Mechanism of actions ....................................................................................... 37
1.6.1. Membrane-active peptides .................................................................... 37
1.6.2. Non-membrane targeting mode of actions ............................................ 43
1.6.3. Mechanism of bacterial AMPs resistance ............................................. 51
1.7. AMPs as novel antimicrobial agents ................................................................ 55
1.7.1. Cathelicidins .......................................................................................... 56
1.7.2. Magainins .............................................................................................. 58
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1.7.3. Prophenins ............................................................................................. 59
1.7.4. Defensins ............................................................................................... 60
1.7.5. Protegrins .............................................................................................. 62
1.8. Designing novel antimicrobial peptides ........................................................... 63
1.9. Justification and objective of the study ............................................................ 66

CHAPTER 2: MATERIALS & METHODS .................................................................. 68
2.1. Isolates collection and identification ................................................................ 69
2.1.1.Hemolytic pattern on blood agar ............................................................. 69
2.1.2.Gram stain ............................................................................................... 70
2.1.3.Catalase test ............................................................................................. 70
2.1.4.Ethylhydrocupreine hydrochloride (optochin) susceptibility test ........... 71
2.1.5.Bile solubility .......................................................................................... 71
2.2. Antibiotic susceptibility testing ........................................................................ 72
2.3. Multiplex Polymerase Chain Reaction (PCR) serotyping ................................ 73
2.3.1.DNA extraction ....................................................................................... 73
2.3.2.Multiplex PCR scheme ............................................................................ 74
2.4. Peptides design ................................................................................................. 78
2.4.1.Selection of lead peptides ........................................................................ 78
2.4.2.Design of synthetic AMPs ....................................................................... 80
2.4.3.Redesigning synthetic antimicrobial peptides with improved antibacterial
activity and cell toxicity .......................................................................... 80
2.4.4.Peptides physicochemical analytical tools .............................................. 85
2.4.5.Peptide synthesis and testing ................................................................... 85
2.5. In vitro assessment of peptides ......................................................................... 86
2.5.1.Bacterial cultures and assay medium ...................................................... 86
2.5.2.Broth microdilution assay ....................................................................... 86
2.5.3.Bacterial killing assay.............................................................................. 87
2.5.4.Synergism study ...................................................................................... 88
2.5.5.Transmission electron microscopy .......................................................... 89
2.5.6.Hemolytic activity ................................................................................... 90
2.5.7.Cytotoxicity against human cell lines ..................................................... 90
2.6. In vivo assessment of peptides .......................................................................... 92
2.6.1.Animal maintenance and handling .......................................................... 92
2.6.2.Injection/administration procedures ........................................................ 92
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