Interactions of Cationic Antimicrobial Peptides with Bacterial Membranes and Biofilms

152 Pages·2012·3.45 MB·English

Interactions of Cationic Antimicrobial Peptides with
Bacterial Membranes and Biofilms
by
Lois Menglu Yin
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Biochemistry
University of Toronto
© Copyright by Lois Menglu Yin 2012
Interactions of Cationic Antimicrobial Peptides with Bacterial
Membranes and Biofilms
Lois Menglu Yin
Master of Science
Department of Biochemistry
University of Toronto
2012
Abstract
Cationic antimicrobial peptides (CAPs) offer a viable alternative to conventional antibiotics as
they physically disrupt the bacterial membranes, leading to cell lysis and death. However,
colonized bacteria often form “biofilms” – characterized by the overproduction of
exopolysaccharides - that restrict the penetration of antibiotics; successful antimicrobial agents
must evade this exopolysaccharide ‘matrix’ to reach the bacterial membrane. Since the
Pseudomonas aeruginosa biofilm alginate traps CAPs by forming peptide-alginate complexes,
the aim of this thesis is to better understand the mechanisms of interaction of CAPs with
bacterial membranes and biofilm alginate. Using a series of CAPs designed in our lab derived
from the sequence KKKKKK-AAFAAWAAFAA-NH2, we found that hydrophobicity, charge
distribution, and amino acid composition of CAPs play important roles in their membrane
disruptive power, bioactivities, alginate-binding and alginate-diffusion abilities. These findings
suggest routes to an optimal balance of factors in CAP design to allow both biofilm penetration
and bacterial membrane destruction.

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Table of Contents

Abstract .......................................................................................................................................... ii
Table of Contents ......................................................................................................................... iii
List of Figures .............................................................................................................................. vii
List of Tables ................................................................................................................................ xi
List of Appendices ....................................................................................................................... xii
List of Abbreviations ................................................................................................................. xiii

Chapter 1 Introduction..................................................................................................................1
1.1 BIOFILMS ...........................................................................................................................2
1.1.1 Formation of biofilms ..............................................................................................2
1.1.2 Environmental and medical problems caused by biofilms ......................................4
1.1.3 Pseudomonas aeruginosa ........................................................................................5
1.1.4 Biofilm exopolysaccharides .....................................................................................6
1.1.5 Alginate ....................................................................................................................7
1.1.6 Mechanisms of biofilm resistance to antimicrobial agents ......................................9
1.1.7 Conventional antibiotics and current treatments for P. aeruginosa infections ......11
1.2 ANTIMICROBIAL PEPTIDES ........................................................................................13
1.2.1 Natural Antimicrobial Peptides..............................................................................13
1.2.2 Mechanisms of Action ...........................................................................................15
1.2.3 De novo Design of Novel CAPs ............................................................................17
1.3 PEPTIDE-MEMBRANE INTERACTIONS .....................................................................22
1.3.1 Composition of Bacterial vs. Mammalian Membranes .........................................22
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1.3.2 Basis for selectivity of CAPs .................................................................................22
1.3.3 Toxicity Threshold of CAPs ..................................................................................24
1.3.4 Effect of Cholesterol in CAP Selectivity ...............................................................26
1.4 PEPTIDE-ALGINATE INTERACTIONS ........................................................................26
1.4.1 Helical Induction of CAPs in Alginate ..................................................................26
1.4.2 Factors that influence peptide-alginate interactions. .............................................28
1.4.2.1 Peptide Concentration .............................................................................28
1.4.2.2 M- vs. G-blocks of Alginate .....................................................................29
1.4.2.3 O-Acetylation of Alginate ........................................................................30
1.4.2.4 Length of Glycan Chain ...........................................................................31
1.5 OVERALL GOALS OF THE THESIS .............................................................................32
Chapter 2 Antimicrobial Activity of Designed Novel CAPs ....................................................34
2.1 INTRODUCTION .............................................................................................................35
2.2 MATERIAL AND METHODS .........................................................................................36
2.2.1 Peptide Synthesis and Purification.........................................................................36
2.2.2 Circular Dichroism.................................................................................................37
2.2.3 Minimal Inhibitory Concentration (MIC) Assay ...................................................38
2.2.4 Time Course Study of Killing ................................................................................38
2.2.5 Purification of Alginate..........................................................................................39
2.2.6 Minimal Inhibitory Concentration in the Presence of Alginate .............................39
2.2.7 Statistical Analysis .................................................................................................39
2.3 RESULTS AND DISCUSSION ........................................................................................40
2.3.1 Secondary Structure of CAPs ................................................................................40
2.3.2 Antimicrobial Activity of Novel CAPs against Various Bacterial Strains ............42
2.3.3 Time Course of Bacteria Killing ............................................................................44
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2.3.4 Alginate Reduces the Activity of CAPs.................................................................46
2.4 SUMMARY .......................................................................................................................47
Chapter 3 Peptide-Membrane Interactions...............................................................................48
3.1 INTRODUCTION .............................................................................................................49
3.2 MATERIAL AND METHODS .........................................................................................50
3.2.1 Peptide Synthesis ...................................................................................................50
3.2.2 Circular Dichroism.................................................................................................50
3.2.3 SDS-PAGE ............................................................................................................51
3.2.4 Computational Modeling of CAPs.........................................................................51
3.2.5 Liposome Preparation ............................................................................................51
3.2.6 Atomic Force Microscopy (AFM) .........................................................................52
3.2.7 Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-
FTIR)......................................................................................................................53
3.2.8 Antibacterial Activity.............................................................................................54
3.2.9 Hemolytic Activity.................................................................................................55
3.3 RESULTS AND DISCUSSION ........................................................................................56
3.3.1 Helix Induction of CAPs in SDS Micelles ............................................................56
3.3.2 CAPs Run as Dimers on SDS-PAGE ....................................................................57
3.3.3 CAP Interactions with Model Bacterial Membranes .............................................59
3.3.4 Positive Charges on Both CAP Termini Minimize Aggregation in Bacterial
Membrane-Mimetic Models ..................................................................................62
3.3.5 Antimicrobial Activity ...........................................................................................64
3.3.6 Interaction of Peptides and Mammalian Membrane Lipids ...................................65
3.3.7 Hemolytic Activity.................................................................................................68
3.4 SUMMARY .......................................................................................................................71
Chapter 4 Peptide-Alginate Interactions ...................................................................................72
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4.1 INTRODUCTION .............................................................................................................73
4.2 MATERIAL AND METHODS .........................................................................................74
4.2.1 Peptide Synthesis ...................................................................................................74
4.2.2 Purification of Alginate..........................................................................................74
4.2.3 Circular Dichroism.................................................................................................74
4.2.4 Tryptophan Fluorescence .......................................................................................75
4.2.5 Alginate-Peptide Binding Affinity Assay ..............................................................75
4.2.6 Fluorophore Labeling.............................................................................................76
4.2.7 Antimicrobial Activity Assay – Minimal Inhibitory Concentration ......................77
4.2.8 Statistical Analysis .................................................................................................77
4.2.9 Penetration of Peptides into Alginate Beads – Laser Scanning Confocal
Microscopy ............................................................................................................77
4.3 RESULTS AND DISCUSSION ........................................................................................78
4.3.1 Titration of Peptides with Alginate ........................................................................78
4.3.2 Tryptophan Fluorescence Blue-Shifts of CAPs in Alginate ..................................80
4.3.3 Alginate-Binding Affinity Assay ...........................................................................85
4.3.4 Diffusion of Peptides into Alginate Beads.............................................................88
4.4 SUMMARY .......................................................................................................................91
Chapter 5 Conclusions and Implications ...................................................................................92

References .....................................................................................................................................98
Appendix .....................................................................................................................................125
Copyright Acknowledgements ..................................................................................................137

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List of Figures

Figure 1-1. Schematic diagram of mushroom-like P. aeruginosa biofilm maturation. .................. 3
Figure 1-2. Formation of a flat P. aeruginosa biofilm. .................................................................. 4
Figure 1-3. Composition and structure of alginate. ........................................................................ 8
Figure 1-4. Mechanisms of biofilm resistance to antimicrobial agents. ....................................... 10
Figure 1-5. Classes of antimicrobial peptides. ............................................................................. 14
Figure 1-6. Mechanisms proposed for cationic antimicrobial peptides (CAP) activity at the site of
the bacterial membrane. ................................................................................................................ 17
Figure 1-7. Schematic representation of natural vs. de novo designed CAPs. ............................ 18
Figure 1-8. Blue shifts (nm) in wavelength maxima emission (Δλ ) of Trp fluorescence upon
max
exposure of selected peptides to large unilamellar vesicles (LUVs) parepared from bacterial lipid
mixtures (LUV-bact) or mammalian red blood cell mixtures (LUV-RBC (outer)). .................... 24
Figure 1-9. Blue shifts (nm), given as Δλmax = Δλmax(aqueous) – Δλmax(LUV), of selected
CAPs to freshly prepared anionic and zwitterionic large unilamellar vesicles (LUV) ................. 25
Figure 1-10. Helical induction of secondary structures in peptides by alginate studied by CD
spectroscopy. ................................................................................................................................. 27
Figure 1-11. Circular dichroism spectra of the X = Phe peptide at 30 µM (dashed line) or 90 µM
(solid line) in 0.2 mg/mL alginate solution in Tris-HCl buffer at pH 7.0. .................................... 29
Figure 1-12. CD spectra of CAP KK-AAAFAAAAAFAAWAAFAAA-KKKK in alginate and in
oligo-D-mannuronate (Alg ) and oligo-L-guluronate (Alg ). ............................................... 30
M-M G-G
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Figure 1-13. % ANS fluorescence emission at increasing concentrations of non-O-acetylated
kelp alginate (- O-acetyl), O-acetylated P. aeruginosa alginate (+ O-acetyl) and the CAP (X =
Phe). .............................................................................................................................................. 31
Figure 1-14. Helical content of CAP KK-AAAFAAAAAFAAWAAFAAA-KKKK at 222 nm of
molar ellipticity as a function of increasing alginate degree of polymerization. .......................... 32
Figure 2-1. Circular dichroism spectra of designed CAPs in aqueous and SDS micelle
environments. ................................................................................................................................ 41
Figure 2-2. Killing potential of designed antimicrobial peptides. ............................................... 43
Figure 2-3. Time course study of antimicrobial activity of peptides against P. aeruginosa (PAO1
strain). ........................................................................................................................................... 45
Figure 2-4. Killing potential of designed antimicrobial peptides in the presence of alginate. ..... 47
Figure 3-1. A photograph of the FTIR set-up. .............................................................................. 54
Figure 3-2. Circular dichroism spectra of CAPs in aqueous solution and SDS micelles. ........... 57
Figure 3-3. (A) SDS -PAGE (silver stain) analysis and (B) representative structural models of
CAP antiparallel dimers. ............................................................................................................... 59
Figure 3-4. AFM topography images of 6k-f17 (a-c) and 6K-F17-4L (d-f) at various
8,11,13,16
concentrations in bacterial membrane lipid mimics. .................................................................... 60
Figure 3-5. Effects of synthetic CAPs on topography images obtained from AFM experiments
performed in tandem with ATR-FTIR measurements in a bacterial model lipid bilayer (3:1
POPE/DOPG). .............................................................................................................................. 62
Figure 3-6. Effects of CAPs on topography images obtained from AFM experiments performed
in tandem with ATR-FTIR measurements in a bacterial model lipid bilayer (3:1 POPE/DOPG).
....................................................................................................................................................... 64
viii
Figure 3-7. Effects of synthetic CAPs on topography images obtained from AFM experiments
performed in tandem with ATR-FTIR measurements in a mammalian model lipid bilayer (1:1:1
DOPC/DSPC/cholesterol). ............................................................................................................ 67
Figure 3-8. Effects of synthetic CAPs on topography images obtained from AFM experiments
performed in tandem with ATR-FTIR measurements in a mammalian model lipid bilayer (1:1:1
DOPC/DSPC/cholesterol). ............................................................................................................ 68
Figure 3-9. A typical hemolysis assay. ......................................................................................... 69
Figure 3-10. Hemolytic Activities of the designed CAPs............................................................. 70
Figure 4-1. Schematic illustration of the alginate affinity binding assay. ................................... 76
Figure 4-2. Schematic illustration of the assay to measure the penetration of peptides into
alginate beads. ............................................................................................................................... 78
Figure 4-3. Circular dichroism spectra of the representative peptide 6K-F17-4L (20 μM)
8,11,13,16
titrated with increments in alginate concentration (0 to 0.6 mg/mL). .......................................... 79
Figure 4-4. Normalized Trp fluorescence emission maxima of the peptides (Table 4-1) in
aqueous solution (solid lines) and in alginate (dotted lines). ........................................................ 81
Figure 4-5. The correlation between the alginate-induced blue shift (nm) and the core segment
hydrophobicity of the peptides (Table 4-1). ................................................................................. 84
Figure 4-6. Structural model of the peptide 6K-F17-2L in a β-strand conformation. ........... 85
11,13
Figure 4-7. The BCA analysis of the percentage of peptides bound to alginate separated from the
free peptides 6K-F17-3L (red) and 6K-F17-2L (black) as a function of alginate
11,13,16 8,16
concentration. ................................................................................................................................ 86
Figure 4-8. The distribution of the dissociation constant, K , of each of the CAPs (listed in Table
d
2-1) binding to alginate. ................................................................................................................ 87
Figure 4-9. Schematic illustration of two peptides with high K values. ..................................... 87
d
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Figure 4-10. Helicity detected by CD (A) and the bacterial inhibition curves measured by the
MIC assay (B) of peptide 6k-a17-f9 (blue) and its TAMRA-labelled analog TAMRA-6k-a17-f9
(red). .............................................................................................................................................. 89
Figure 4-11. Confocal laser scanning micrographs obtained of the interactions and extent of
diffusive penetration of TAMRA-labelled CAPs into alginate beads. ......................................... 90
Figure 5-1. Proposed mechanisms of action of CAPs in bacterial membranes vs. mammalian
membranes. ................................................................................................................................... 96

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