Novel Antimicrobial Peptides in Alligator and Crocodile

108 Pages·2015·1.69 MB·English

by  Stephanie Barksdale

NOVEL ANTIMICROBIAL PEPTIDES IN ALLIGATOR AND CROCODILE
by

Stephanie Barksdale
A Thesis
Submitted to the
Graduate Faculty
of
George Mason University
in Partial Fulfillment of
The Requirements for the Degree
of
Master of Science
Biology

Committee:

Dr. Monique van Hoek, Thesis Director
Dr. Barney Bishop, Committee Member
Dr. Sergei Popov, Committee Member
Dr. James Willett, Director, School of
Systems Biology
Dr. Donna M. Fox, Associate Dean, Student
Affairs & Special Programs, College of
Science
Dr. Peggy Agouris, Dean, College of
Science
Date: Fall Semester 2015
George Mason University
Fairfax, VA
Novel Antimicrobial Peptides in Alligator and Crocodile
A Thesis submitted in partial fulfillment of the requirements for the degree of Master of
Science at George Mason University
by
Stephanie Barksdale
Bachelor of Science
George Mason University, 2015
Director: Monique van Hoek, Associate Professor
School of Systems Biology
Fall Semester 2015
George Mason University
Fairfax, VA
This work is licensed under a creative commons
attribution-noderivs 3.0 unported license.

ii
DEDICATION
This is dedicated to my husband Nick, for his love and support, and to my mother, for
instilling in me a love of learning.
iii
ACKNOWLEDGEMENTS
I would like to thank my family, friends, and lab mates for their support. My husband
Nick supported me through long hours and hard work. My sisters Jessica and Rachael
and my parents have encouraged me to push myself. My PI Dr. van Hoek has mentored
me for many years and has helped me grow as a researcher, an employee, and a person.
All of my lab mates helped me get through difficult times and difficult problems. And
thanks to the Dr. Bishop and his lab for all of the collaboration and advice from the other
side of biochemistry.

iv
TABLE OF CONTENTS
Page
List of Tables ..................................................................................................................... vi
List of Figures ................................................................................................................... vii
List of Equations .............................................................................................................. viii
List of Abbreviations and/or Symbols ............................................................................... ix
Abstract .............................................................................................................................. xi
Chapter 1: Introduction ....................................................................................................... 1
Antimicrobial Peptides .................................................................................................... 1
Order Crocodilia .............................................................................................................. 3
Alligator mississippiensis (American Alligator) ......................................................... 4
Crocodylus siamensis (Siamese Crocodile) ................................................................ 4
Infecting and Commensal Bacteria of Alligators and Crocodiles ................................... 5
Innate Immunity of Crocodilians .................................................................................... 8
Chapter 2: Hepcidin of the Siamese crocodile .................................................................. 15
Chapter 3: Apolipoprotein-derived peptides of the American Alligator .......................... 36
Chapter 4: Cathelicidin of the American Alligator ........................................................... 56
Chapter 5: Conclusion....................................................................................................... 76
References ......................................................................................................................... 80

v
LIST OF TABLES
Table Page
Table 1 Siamese crocodile hepcidin sequence and properties…………………………...19
Table 2 Reduction conditions and rate equations for antimicrobial assays……………...21
Table 3 Amino acid sequence conservation in hepcidins………………………………..24
Table 4 Reported antimicrobial and antifungal activity of various hepcidins…………...27
Table 5 Antimicrobial activity of Siamese crocodile hepcidin……...…………………...28
Table 6 Apo5 and Apo6 sequences and physio-chemical properties……………………40
Table 7 Reduction conditions and rate equations for antimicrobial assays……………...41
Table 8 Percent secondary structure contribution of Apo5 and Apo6…………………...46
Table 9 Antimicrobial activity of Apo5 and Apo6……………………………………....47
Table 10 Genes from the A. mississippiensis genome annotated as cathelicidins.............63
Table 11 Identification of alligator cathelicidin genes......................................................65
Table 12 Known OH-CATH-like antimicrobial peptides..................................................66
Table 13 Sequence and physico-chemical properties of AM-CATH28............................67
Table 14 Antimicrobial activity of AM-CATH28.............................................................68
Table 15 Percent secondary structure contribution of AM-CATH28................................71

vi
LIST OF FIGURES
Figure Page
Figure 1 Classes of AMP secondary structures...................................................................2
Figure 2 Blood cells from Alligator mississippiensis........................................................10
Figure 3 Conserved disulfide bond patterns in hepcidins..................................................16
Figure 4 Antimicrobial activity of the Siamese crocodile hepcidin..................................29
Figure 5 CD spectra of the Siamese crocodile hepcidin....................................................31
Figure 6 Bioinformatical analysis of Apo5 and Apo6.......................................................44
Figure 7 CD spectra of Apo5 and Apo6............................................................................45
Figure 8 Graph of antimicrobial activity of Apo5 and Apo6.............................................49
Figure 9 Membrane permeabilization activity by Apo5 and Apo6..................... .............50
Figure 10 Membrane depolarization by Apo5 and Apo6..................................................51
Figure 11 Cytotoxicity of Apo5 and Apo6........................................................................52
Figure 12 Helical wheels of AM-CATH28 and AM-CATH.............................................67
Figure 13 Structural prediction of AM-CATH28..............................................................70
Figure 14 Membrane permeabilization activity by AM-CATH28....................................72
Figure 15 Cytotoxicity of AM-CATH28...........................................................................72

vii
LIST OF EQUATIONS
Equation Page
Equation 1 Growth rate equation for P. aeruginosa ATCC 9027.....................................21
Equation 2 Growth rate equation for S. aureus ATCC 25923...........................................21
Equation 3 Growth rate equation for E. coli ATCC 51659...............................................21
Equation 4 Growth rate equation for P. aeruginosa PAO1...............................................41
Equation 5 Growth rate equation for S. aureus ATCC 33592...........................................41
Equation 6 Growth rate equation for A. baumannii ATCC BAA-1794............................41
viii
LIST OF ABBREVIATIONS
Amino Acid........................................................................................................................aa
Cationic Antimicrobial Peptide..................................................................................CAMP
Liver Expressed Antimicrobial Peptide-1................................................................LEAP-1
Lipopolysaccharide.........................................................................................................LPS
Trifluoroethanol..............................................................................................................TFE
Sodium Dodecyl Sulfate.................................................................................................SDS
Phosphate Buffer...............................................................................................................PB
Phosphate-Buffered Saline.............................................................................................PBS
Colony-Forming Units...................................................................................................CFU
Circular Dichroism...........................................................................................................CD
Enterohemorrhagic E. coli...........................................................................................EHEC
Multi-Drug Resistant....................................................................................................MDR
Reverse Phase High-Performance Liquid Chromatography.................................RP-HPLC
Electrospray Ionatization Mass Spectrometry..........................................................ESI-MS
Optical Density.................................................................................................................OD
Centers for Disease Control and Prevention..................................................................CDC
Pathogen-associated molecular pattern.......................................................................PAMP
Methicillin-resistant S. aureus....................................................................................MRSA
Deoxyribonucleic acid...................................................................................................DNA
Messenger ribonucleic acid........................................................................................mRNA
National Center for Biotechnology Information..........................................................NCBI
Expressed sequence tag..................................................................................................EST
American Type Culture Collection.............................................................................ATCC
Sheep Myeloid Antimicrobial Protein-29..............................................................SMAP-29
Basic Local Alignment Search Tool.........................................................................BLAST
European Molecular Biology Laboratory...................................................................EMBL
genInfo identifier...............................................................................................................GI
Antimicrobial Peptide Database 2...............................................................................APD2
Molecular weight............................................................................................................MW
Trifluoroacetic acid........................................................................................................TFA
Weight/volume.................................................................................................................w/v
Volume/volume................................................................................................................v/v
Confidence interval............................................................................................................CI
Base pair.............................................................................................................................bp
Minimal inhibitory concentration...................................................................................MIC
Minimal bactericidal concentration..............................................................................MBC
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