Antimicrobial Peptides - Edinburgh Research Archive - University of

171 Pages·2009·23.57 MB·English

Antimicrobial Peptides – Structure, Function and
Resistance

Thomas Vargues
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Doctor of Philosophy

The University of Edinburgh
2009
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Declaration
Declaration

I, Thomas Vargues, hereby certify that this thesis has been composed by myself, and
that it is a record of my work, and that it ahs not been accepted in partial or complete
fulfilment of any other degree or professional qualification.

Thomas Vargues
University of Edinburgh
2009

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Acknowledgements
Acknowledgements

First of all I would like to thank Dr Dominic Campopiano for giving me the
opportunity to do a PhD in this very interesting field. His constant supervision and his
scientific “flair” kept me motivated and focused.
I would like to thanks Prof John Govan for allowing me to study in his
laboratory at little France and for sharing very interesting discussions in this research
field.
I have to thank Dr Derek Macmillan from University College London and Dr
Dusan Uhrin for their help and technical support in research fields I was not familiar
with.
A special thank for Emily Seo who helped me a lot when things were not at
their best. I would like to thank Dave Clarke and Gareth Morrison for their team
support but also Marine, Jon and Karin for all the help provided and all members of
Lab 229, past and present.

I don’t know where to start to thank my cousin Julien Valton for priceless help
and “positive thinking” towards this thesis. I am also grateful to Gregory Vernier for
his advices and experience regarding many topics linked to this study. I really enjoyed
such endless and helpless discussions across the ocean. We will meet for sure around
a good “Whiskass chaton”. I would also like to thank Carole Mathevon for all the
advices provided and nice chats over the phone.

An enormous thank for all my family for things only they know. Florent,
Guillaume, Augustin, Mum, Dad and Mam. I think my grandfather would have been
proud of this also…

Finally, a very special thank will go for a tricky “weegie” girl, Aileen who
gave me joy and hope for these last two years. Your humour and constant support
were priceless to me.

And thanks for the one I forgot……….

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Abstract
Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play
important roles in their defence against microbial infection. Beta defensins are small
(3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a
range of microbes and also act as chemoattractants of important immunomodulatory
cells. To generate highly pure peptides for structural and functional studies, we
developed a method to prepare recombinant human beta defensin-2 (HBD2). The
HBD2 gene was synthesised by recursive PCR with codons optimised for expression
in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged
ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H
NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was
folded and possessed the correct β-defensin disulfide bond topology. The recombinant
HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was
also a chemoattractant against HEK293 cells expressing the chemokine receptor
CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future
structural studies. Since defensins are thought to interact with bacterial membranes we
also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance,
SPR, Biacore). We observed different binding to artificial model membranes
containing either E. coli Kdo -lipid A or phospholipids.
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Bacterial resistance to AMPs has been linked to the covalent modification of
the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This
neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of
cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia
displays extremely high resistance to AMPs and other antibiotics and the Ara4N
pathway appears to be essential. To explore this further we expressed recombinant
forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway.
Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we
tested its ability to bind amino acid substrates. We investigated the binding of
inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against
Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed
membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein
behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed
to complement E. coli knock-out strains encoding the equivalent flippase proteins
ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.
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Abbreviations
Abbreviations

Aa Amino acid(s)
AMP Antimicrobial peptide
APS Ammonium persulfate
ArnT L-Ara4N transferase
Bcc Burkholderia cepacia complex
CF Cystic fibrosis
CFTR Cystic fibrosis transmembrane conductance regulator
cfu Colony forming units
Da Dalton
DDM n-Dodecyl-β-maltoside
DDT Dithiothreitol
EDTA Ethylenediaminetetracetic acid
ESI-MS Electrospray ionisation mass spectroscopy
FPLC Fast protein liquid chromatography
FT-ICR Fourier transform ion cyclotron resonance
HBD1 Human β-defensin-1
HBD2 Human β-defensin-2
HBD3 Human β-defensin-3
HBD4 Human β-defensin-4
g Gravity (centrifugal force)
HEPES N-(2-Hydroxyethyl)piperazine-N`-(2-ethanesulfonic acid)
IMAC Immobilized metal affinity chromatography
IPTG Isopropyl-1-thio-β−D-galactopyranoside
ISA Isosensitest agar
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Abbreviations

ISB Isosensitest broth
kDa Kilo daltons
Kdo 3-deoxy-D-manno-oct-2-ulosonic acid
Ko D-glycero-α-D-talo-oct-2-ulosonic acid
L-Ara4N 4-amino-4-deoxy-β-L-arabinose
LB Luria Bertani
LC-MS Liquid chromatography mass spectrometry
LD Lethal dose

LPS Lipopolysaccharide
MIC Minimum inhibitory concentration
PAGE Polyacrylamide gel electrophoresis
PLP Pyridoxal 5’-phosphate
PMP Pyridoxamine phosphate
PBS Phosphate buffered solution
PCR Polymerase chain reaction
PET Phospoethanolamine
PMB Polymyxin B
SDS Sodium dodecylsulfate
SPR Surface plasmon resonance
SPPS Solid Phase Peptide Synthesis
TB Terrific broth
TCEP Tris carboxyethylphosphine
TFA Trifluoroacetic acid
Tris Tris [hydroxymethyl] aminomethane
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Abbreviations
Ugd UDP-glucose dehydrogenase
UV-Vis Ultraviolet- visible spectroscopy
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Table of Contents
Table of Contents

Declaration ii
Acknowledgements iii
Abstract iv
Abbreviations v
Table of contents viii
List of Figures xiv
List of Tables xviii

Chapter 1: Defensins - Multifaceted Peptides of the Innate and Adaptive
Immune System 1
1.1 The Expanding Diversity of The Antimicrobial Peptide Family 2
1.2 Defensins 3
1.2.1 Structural Classification 3
1.2.2 From the Innate to the Adaptive immune system 5
1.2.3 Antiviral Properties 7
1.2.4 Additional Biological Functions 8
1.3 The Human β-Defensins 9
1.3.1 Discovery and Isolation 9
1.3.2 Structural Characteristics 11
1.3.3 Expression and Gene Regulation 12
1.3.4 Genes and Post-Translational Modifications 13
1.3.5 Antimicrobial Activity 14
1.3.6 Structure Activity Relationships 15
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Table of Contents
1.3.7 Mode of Production of Defensins 20

Chapter 2: Expression, Purification and Characterisation of the Mature
Recombinant Human ββββ-Defensin 2 (HBD2) 23
2.1 Cloning of HBD2 24
2.2 Overexpression and Purification of KSI-HBD2 Fusion Protein 25
2.3 CNBr Cleavage of KSI-HBD2 Fusion Protein and Purification of HBD2 Using a
Fast Protein Chromatography Liquid (FPLC) System 26
2.4 Expression and Purification of HBD2 Using the pET-28a/HBD2 Plasmid and an
HPLC System 28
2.5 Refolding of the Recombinant Defensin 29
2.6 Determination of Disulfide Connectivity for HBD2 32
2.7 Structural Characterisation of Mature (cid:1)-Defensins 34
2.8 Expression, Purification and Characterisation of the 15N Labelled-HBD2 36
2.9 Antimicrobial Properties of HBD2 41
2.10 Surface Plasmon Resonance (SPR) Binding Studies of HBD2 with Biological
Membrane Models 42

Chapter 3: Comparison of Synthetic and Recombinant Production of Defensins
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3.1 Solid-Phase Peptide Synthesis (attempt) of HBD1 47
3.2 Recombinant Expression and Purification of HBD1 49
3.3 Conclusions (Chapter 2 & 3) 52
3.4 References Chapter 1, 2 and 3 55

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Chapter 4: Into the resistance 66
4.1 Bacteria Fight Back 67
4.2 LPS and Lipid A Biosynthesis in Gram-Negative Bacteria 69
4.3 Lipid A Modifications in Gram-Negative Bacteria 72
4.4 Cystic Fibrosis (CF) and The Burkholderia Cepacia Complex 76
4.5 The Biosynthetic Pathway of Aminoarabinose (L-Ara4N) Modification of Lipid
A: The Burkholderia Cenocepacia Case 78
4.6 Structure of B. Cenocepacia Lipopolysaccharide 82
4.7 Aims 84

Chapter 5: Characterisation of Enzymes Involved in Ara4N biosynthesis in
Burkholderia cenocepacia 85
5.1 Analysis of the arn Operon in B. cenocepacia J2315 86
5.2 Cloning, Expression and Purification of ArnB from B. cenocepacia 92
5.3 Spectroscopic Characterisation of ArnB 95
5.4 Assays of ArnB with Potential Inhibitors 99
5.5 Antimicrobial Assays of D-Cycloserine and Polymyxin B 104
5.6 Cloning, Expression and Purification of ArnG (BCAL 1930) 108
5.7 Complementation assay of ArnG in E. coli knock-out strains 110
5.8 Conclusions and Perspectives 114
5.9 References Chapter 4 & 5 117

Chapter 6: Material and Methods 127
6.1 Material and Methods for Chapters 2 and 3 128
6.1.1 General Materials 128
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