Investigation of the interaction of antimicrobial peptides with lipids and lipid membranes 1

4.3.5 Determination of aggregation number of LPS using FITC-LPS and Triton X-100 75 CHAPTER 5 INVESTIGATION OF THE BINDING OF A NOVEL ANTIMICROBIAL PEPTIDE V4 TO MEMBRANE MIMICS 5.3.6 Co

INVESTIGATION OF THE INTERACTION OF

ANTIMICROBIAL PEPTIDES WITH LIPIDS AND LIPID

This work was performed in the biophysical fluorescence laboratory, Department of Chemistry, National University of Singapore under the supervision of Asst Prof Thorsten Wohland

The results have been partly published in:

Yu, L., Ding, J L, Ho, B and Wohland, T Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: An amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and

antimicrobial activity Biochimica et Biophysica Acta (BBA) – Biomembranes 1716,

Acknowledgements

A thesis like this, involving different and varied fields, cannot be undertaken without the help of many people I would like to take this opportunity to thank the persons who especially provide help in my study

First, I would like to express my deepest gratitude to my supervisor Asst Prof Thorsten Wohland for giving me the opportunity to work in such an interesting field of antimicrobial peptides I would like to thank him for always offering the guidance, support and assistance during the course of this work

I would like to thank Prof Ding Leak Ling and Ho Bow, for giving me many valuable and scientific suggestions and constructive discussion on the project, which is a great help to me

I would like to thank Assoc Prof Feng Si Shen for his kind help of providing instrument

of Langmuir film balance for my study

I am grateful to all the members of biophysical fluorescence laboratory for their enthusiastic help and support

And last but not least I would like to thank my parents and my husband for their kind understanding, continuous support and unconditional love all these years

2.1 Biological activities of antimicrobial peptides 7

2.3 Structural features of antimicrobial peptides 17

2.7 Therapeutic potential of antimicrobial peptides 36

3.1 Techniques used in the study of antimicrobial peptides 393.2 Fluorescence Correlation Spectroscopy (FCS) 393.2.1 Principle of Fluorescence Correlation Spectroscopy (FCS) 39

3.3.3 Realization of the surface pressure measurement 52

CHAPTER 4 DETERMINATION OF CRITICAL MICELLE

CONCENTRATIONS AND AGGREGATION NUMBERS OF

DETERGENT AND LPS

56

4.3.1.1 Titration of micelles with fluorophore 614.3.1.2 Disaggregation of FITC-LPS with a detergent 654.3.2 Calculation of titrating a solution of aggregates with a

fluorescent probe

664.3.3 Determination of aggregation number of C12E9 using R18 694.3.4 Determination of aggregation number of LPS using R18 74

4.3.5 Determination of aggregation number of LPS using FITC-LPS

and Triton X-100

75

CHAPTER 5 INVESTIGATION OF THE BINDING OF A NOVEL

ANTIMICROBIAL PEPTIDE V4 TO MEMBRANE MIMICS

5.3.6 Comparison of V4-TMR binding to LPS, lipid A and PC 905.3.7 Binding to V4-TMR to SUVs of pure lipids 925.3.8 Interaction of V4-TMR with mixed lipid SUVs 94

5.4.1 V4-TMR aggregates and is strongly quenched in PBS 95

5.4.3 V4-TMR functions via hydrophobic and electrostatic forces 995.4.4 Saturation of lipids affects the binding with V4-TMR 100

CHAPTER 6 INVESTIGATION OF THE MECHANISMS OF

ANTIMICROBIAL PEPTIDES INTERACTING WITH MEMBRANES

101

6.3.1 Antimicrobial peptides induce leakage of rhodamine 6G

entrapped POPG LUVs

105

6.3.2 Antimicrobial peptides interact with Rho-PE labeled POPG

LUVs

1136.3.3 Antimicrobial peptides induce leakage of rhodamine 6G

entrapped DPPG LUVs

1196.3.4 Antimicrobial peptides interact with Rho-PE labeled DPPG

LUVs

1236.3.5 Visualization of antimicrobial peptides interacting with Rho-

POPG LUVs

131

6.4.2 Comparison of different antimicrobial peptides 136

CHAPTER 7 INTERACTION BETWEEN ANTIMICROBIAL PEPTIDES

AND LIPID MEMBRANE LAYERS

141

7.3.1 Isotherm studies of V4 interacting with POPG and POPC 145

7.3.1.2 Isotherms of mixed lipid/V4 monolayers 1467.3.1.3 Miscibility analysis of monolayers 1487.3.1.4 Stability analysis of monolayers 1517.3.1.5 Compressibility analysis of monolayers 1537.3.2 Penetration studies of antimicrobial peptides interacting with

monolayers

1587.3.2.3 Penetration of V4 into different lipid monolayers 1637.3.2.4 Effect of lipid packing on the penetration of V4 1687.3.3 AFM studies of antimicrobial peptides interacting with lipid

monolayers

176 7.3.4 Insertion of V4 into solid supported bilayer 182

Bibliography 192

Summary

Antimicrobial peptides serve as a significant weapon of organisms to defend against microbial infections Different from conventional antibiotics, antimicrobial peptides mainly target bacterial membranes, induce permeation and eventually lead to bacterial lysis Because it is difficult for bacteria to develop resistance, antimicrobial peptides have been considered a promising drug candidate as a substitute or addition for conventional antibiotics Many native antimicrobial peptides have a broad-spectrum activity against different microorganisms However, due to the lack of selectivity, their application as

therapeutics is limited Therefore de novo design of antimicrobial peptides has become an

interesting method to provide new and efficient drug candidates V4 which is designed based on some sequences of endotoxin-binding host defense proteins has an amphipathic pattern of HBHPHBH (H: hydrophobic; B: basic; P: polar residue) and displays a good combination of high antimicrobial activity, low cytotoxic activity and low hemolytic activity Its greatly enhanced antimicrobial potency is worthy of further investigation

This study investigated the interaction between V4 and different membrane components

to unravel the mechanism of V4 targeting membranes Peptide binding, insertion and membrane permeation were examined The physical property of V4 and the binding affinity for different membrane components, including LPS, lipid A and distinct phospholipids, were studied by fluorescence correlation spectroscopy (FCS) The results show that V4 is a highly hydrophobic peptide, leading to a strong aggregation in solution Only a small percentage of V4 is active At low peptide/lipid ratio, V4 shows a higher

binding affinity for anionic membrane components compared to the zwitterionic lipids This strong interaction of V4 with negatively charged lipids indicates that electrostatic force is a prerequisite for its selective action on bacterial membranes in contrast to mammalian membranes The insertion of V4 into membranes was examined by Langmuir film balance and atomic force microscopy V4 shows higher ability to penetrate into negatively charged membranes than neutral membranes, which confirms the significance

of electrostatic interaction Except insertion, the formation of filaments was also observed, indicating a peptide aggregation At high peptide/lipid ratio, V4 is shown to induce membrane permeation by causing membrane aggregation and disruption The mechanism

of V4 interacting with membrane is also compared to other antimicrobial peptides magainin 2, melittin and polymyxin B

Necessary for the understanding of V4 binding to bacterial membranes is an understanding of the aggregation behavior of the main target, LPS Therefore, the aggregation property of LPS was examined using a new method based on FCS, which allows the determination of aggregation numbers of complexes from the amplitudes of the correlation function This method was evaluated by a well-known detergent C12E9 and showed a good agreement with literature values, which confirmed the feasibility of the method Later the method was applied on LPS to determine the critical micelle concentration and aggregation number

This study should enhance the understanding of the predominant specificity of V4 and the mode of action on membranes The results could contribute to the rational design of

novel antimicrobial peptides and provide useful information to develop new antimicrobial drugs

List of Tables

Table 2.1 Representative antimicrobial peptides with different secondary

structure

12,13

Table 4.1 Number N of particles per observation volume of FITC-LPS

solutions of concentrations between 1 and 50 µM before and after treatment with Triton X-100

76

Table 5.2 Comparison of TMR, V4-TMR, V4-TMR: LPS on coverslip 90Table 5.3 Comparison of interaction of V4-TMR peptide with LPS, lipid A

Table 7.1 Comparison of height differences of POPG monolayers penetrated

by different antimicrobial peptides

176

List of Figures

Figure 2.1 Schematic drawing of gram-positive and gram-negative bacterial

membranes

23

Figure 2.2 Schematic drawing of the mechanisms of antimicrobial peptide

interacting with membranes

Figure 3.2 Schematic drawing of the relationship of ACF and fluorescence signals 45

Figure 3.4 The effect of laser power on the photon count rates and diffusion time 48Figure 3.5 ACFs of rhodamine 6G with different concentrations 49Figure 3.6 Particle number and diffusion time of rhodamine 6G with different

concentrations

49

Figure 3.7 The effect of glycerol on the diffusion time of rhodamine 6G 49Figure 3.8 Molecules on the interface of liquid and air 51Figure 3.9 Wihelmy plate method to measure surface pressure 53

Figure 3.11 Schematic drawing of antimicrobial peptides penetrating into lipid

monolayers

55

Figure 4.3 Calculation of the titration curves (cps vs concentration of probe

Figure 4.5 ACFs taken in the transition region around the CMC 71Figure 4.6 Titration of C12E9 and LPS solutions with R18 73Figure 4.7 Titration of 10 and 20 nM of R18 with increasing concentrations of

LPS (0.25-5 µM)

74

Figure 4.8 Dissolution of FITC-LPS micelles by Triton X-100 76

Figure 5.2 ACFs of 200 nM V4-TMR in PBS, water and 0.05% Troton X-100 and

ACFs of 1 nM rhodamine 6G and 100 nM V4-TMR in DMSO

86

Figure 5.3 ACF of 100 nM V4-TMR with different concentrations of LPS in PBS 88Figure 5.4 Titration of 100 nM V4-TMR with increasing concentrations of LPS 89Figure 5.5 Comparison of ACFs of V4-TMR and complexes of V4-TMR with

LPS, lipid A and PC

91

Figure 5.6 Comparison of V4-TMR binding to different SUVs 93Figure 5.7 Binding of V4-TMR to SUVs of mixed lipid composition 94Figure 6.1 Schematic drawing of the investigation of the mechanisms of

antimicrobial peptides by rhodamine 6G entrapped LUVs and Rho-PE labeled LUVs

102

Figure 6.2 Interaction of antimicrobial peptides with rhodamine 6G entrapped

POPG LUVs

108

Figure 6.3 Comparison of N rho , N vesicle , F 2 and photon count rates of antimicrobial

peptides interacting with rhodamine 6G entrapped POPG LUVs

111

Figure 6.4 Interaction of antimicrobial peptides with Rho-PE labeled POPG LUVs 116Figure 6.5 Aggregation caused by melittin at peptide/lipid ratio of 1:2.67 117

Figure 6.6 Comparison of N app and diffusion time of antimicrobial peptides

interacting with Rho-PE labeled POPG LUVs

118

Figure 6.7 Interaction of antimicrobial peptides with rhodamine 6G entrapped

DPPG LUVs

120

Figure 6.8 Comparison of N rho , N vesicle , F 2 and photon count rates of antimicrobial

peptides interacting with rhodamine 6G entrapped DPPG LUVs

123

Figure 6.9 Interaction of antimicrobial peptides with Rho-PE labeled DPPG LUVs 125

Figure 6.10 Comparison of N app and diffusion time of antimicrobial peptides

interacting with Rho-PE labeled DPPG LUVs

127

Figure 6.11 Confocal images of Rho-PE labeled LUVs in the absence and presence

of antimicrobial peptides

129

Figure 6.12 Antimicrobial peptides interacting with rhodamine 6G entrapped POPG

LUVs with different incubation times

130

Figure 6.13 Antimicrobial peptides interacting with rhodamine 6G entrapped DPPG

LUVs with different incubation times

130

Figure 6.14 Leakage of fluorophore entrapped LUVs (20 µM POPG lipid) was

caused by V4 (10 µM) in less than 10 minutes

132

Figure 7.2 Isotherms of mixed POPG/V4 and POPC/V4 monolayers 148Figure 7.3 Surface pressure of lipid monolayers incorporated with different

Figure 7.7 Kinetics of antimicrobial peptides with different concentrations

penetrating into POPG monolayers

157

Figure 7.8 Comparison of antimicrobial peptides penetrating into POPG

monolayers

157

Figure 7.9 Comparison of melittin, polymyxin B and V4 penetrating into POPG

and POPC monolayers

160

Figure 7.10 Kinetics of melittin and V4 with different concentrations penetrating

into POPC monolayers

permeation

185

CHAPTER 1 INTRODUCTION

Antibiotics are one of the most important discoveries in history and save a large number

of lives and greatly increase the average life expectancy1 They are widely applied to food and clinical uses to control many bacterial infections However the increasing antibiotic resistance, which is caused by extensive and unnecessary clinical use, as well

as the lack of new class of antibiotics make it necessary to look for and develop new antimicrobial drugs1 Emerging potent candidates are antimicrobial peptides Different from conventional antibiotics which either disrupt the bacterial wall synthesis or destroy peptide synthesis, antimicrobial peptides mainly target the bacterial membrane, make it permeable and eventually lead to bacterial death Because it is difficult for bacteria to develop a new membrane system to survive, antimicrobial peptides have been considered

a promising drug candidate as a substitute or addition for conventional antibiotics

Antimicrobial peptides are ancient weapons of organisms to defend against microbial infections, and have evolved 2.6 billion years2 They have been widely found in nature including insects, amphibians, mammals and humans Nowadays the genes of many antimicrobial peptides have been characterized and the expression profiles have been established3 Antimicrobial peptides are an essential component of innate immunity, and are either inducible or constitutive4 They are present on the mucosal surfaces, on the body surface and within the granules of phagocytes within several minutes to hours shortly after a bacterial infection4 This response is much faster than the adaptive immunity in the higher vertebrates which usually needs several days to induce an

immune reaction This rapid response provides organisms the first protection against infections Antimicrobial peptides have the ability to defend against a variety of microorganisms, including gram-positive bacteria, gram-negative bacteria, fungi, protozoa, some tumors and viruses Some antimicrobial peptides are also toxic to mammalian cells while others have the capability of discriminating host cells from microbial cells, which have more therapeutic potential Regardless of the distinct origins, primary sequences, biological activities and secondary structures, most antimicrobial peptides share some common characteristics They are short peptides, consisting of less than 50 amino acids in length Their small size makes chemical synthesis possible for a variety of investigations3 Most antimicrobial peptides are cationic with +2 to +9 charges due to the presence of lysine and/or arginine residues and have amphipathic structures, which spatially separate hydrophobic and hydrophilic amino acids to facilitate the interaction with membranes The charge and the amphipathicity are crucial factors for determining the biological activities of antimicrobial peptides Antimicrobial peptides are believed to perform their functions via a non-specific mechanism by directly targeting bacterial membranes to induce non-receptor-mediated membrane permeation5 They either perturb the membrane to form pores/channels and permit a leakage of cellular components as well as dissipating the electrical potential of the membranes or disrupt the membrane in a detergent like manner6 The interaction between antimicrobial peptides and membranes has been extensively investigated to examine the selectivity of antimicrobial peptides killing bacteria, not harming mammalian cells, and to unravel the mechanism of the action of antimicrobial peptides, which are the keys for the rational

design of novel antimicrobial drugs A more detailed overview of antimicrobial peptides can be found in Chapter 2

Because many natural antimicrobial peptides kill bacteria and are toxic to mammalian cells, few of them are available for medical use An alterative way to develop new drugs

is to design new antimicrobial peptides based on known peptide sequences and structures Screening for high antimicrobial activity, low cytotoxicity and low hemolytic activity can then be used for selection of potential drug candidates V4 is designed based on such a strategy with a pattern of HBHPHBH (H: hydrophobic residue; B: cationic residue; P: polar residue) This peptide showed a specificity 2400-fold greater compared to polymyxin B, which is a well known antimicrobial peptide However, little is known about this novel designed peptide The basis for the high selectivity towards bacterial membranes and the mechanism of V4 targeting membranes are still not understood The greatly enhanced antimicrobial potency of V4 is worthy of further investigation as a potent antimicrobial agent

This thesis will describe the properties of V4, including the physical properties, binding affinity for different components, interaction with different lipid membranes, the ability

to induce membrane permeation and morphology, and possible action mechanism As an artificial antimicrobial peptide, due to the great ability to inhibit gram-negative bacteria

in vivo compared to polymyxin B, the mechanism of V4 is also compared with

polymyxin B The cyclized antimicrobial peptides strongly interact with membranes, however, the action mechanism is not clear Therefore in this study the mechanism of

cyclized peptides, V4 and polymyxin B, is also compared to that of α-helix antimicrobial peptides, magainin 2 and melittin

This thesis will present a more complete picture to show how V4 works as well as the comparison of V4 with other antimicrobial peptides More specifically, the aims of this thesis are:

1) To examine the aggregation property of Lipopolysaccharide (LPS) for further determination of binding of V4

2) To investigate the solubility of V4 in different solvents and find an appropriate working condition

3) To study the binding affinity of V4 for LPS, lipid A which contribute to the outer membrane of negative bacteria, and different phospholipids liposomes which mimic the cytoplasmic membranes to elucidate the membrane activity and specificity of V4

4) To examine the ability of V4 to induce membrane permeation and compare with other antimicrobial peptides magainin 2, melittin and polymyxin B

5) To investigate the interaction of V4 with different lipid monolayers including insertion ability and membrane morphological change, and compare with other antimicrobial peptides

6) To elucidate the possible mechanisms of studied antimicrobial peptides interacting with membranes

The study should enhance the understanding of the predominant specific property of V4 and the mode of action on membranes The study may also provide some evidence for existing mechanism hypothesis about antimicrobial peptides Moreover, the results could contribute to the rational design of novel antimicrobial drugs and provide some useful information to develop new antimicrobial drugs

In this thesis fluorescence correlation spectroscopy, Langmuir film balance and atomic force microscopy are used for investigation Especially fluorescence correlation spectroscopy which is the main technique used in this study is proved to be a useful tool

to study the interaction between antimicrobial peptides and membranes and the action mechanism in relation to membranes Different membrane models are applied in this study, including micelles, small and large unilamellar vesicles (liposomes) as well as monolayers Micelles and small unilamellar vesicles are used for investigation of the binding affinity of V4 and large unilamellar vesicles are used to study membrane permeation Monolayers are applied to investigate the insertion of antimicrobial peptides and their effect on membrane morphology

The organization of this thesis is as follows Chapter 1 of this thesis gives a brief description about the background and objective of this study In chapter 2 a detailed overview of antimicrobial peptides is presented Chapter 3 describes the principle of fluorescence correlation spectroscopy and Langmuir film balance In chapter 4 a method

to measure the aggregation number and critical micelle concentration of micelles is developed The method is evaluated by a known detergent C12E9 and applied to determine

the aggregation of LPS for further study Chapter 5 gives the investigation of the physical properties of V4, binding affinity for different membrane components In chapter 6 a detailed investigation of antimicrobial peptides inducing membrane permeation and possible mechanisms are given Chapter 7 describes the interaction of antimicrobial peptides with membrane layers and the induced membrane morphological change using Langmuir film balance and atomic force microscopy Finally chapter 8 provides the conclusion of this study and the outlook

CHAPTER 2 OVERVIEW OF ANTIMICROBIAL PEPTIDES

2.1 Biological activities of antimicrobial peptides

Antimicrobial peptides have a broad-spectrum of activity against different microorganisms including bacteria, fungi, protozoa, malignant cells and some viruses Some antimicrobial peptides exhibit the ability to defend against several microbial infections, while others show specificity for a particular microorganism There are also some antimicrobial peptides which are not only toxic to microbes but also to mammalian cells The specificity of antimicrobial peptides is believed to be mainly due to the different membrane components of microbes and mammalian cells

Magainins which were first discovered from the skin of the African clawed frog, Xenopus

laevis by Zasloff in 1987 show a broad spectrum of antimicrobial activity against

gram-positive bacteria, gram-negative bacteria, fungi and protozoa, but are not toxic to mammalian cells at the same concentration7-11 Gramicidin S which was first isolated

from bacillus brevis by Gause and Brazhnikova has considerable specificity for

gram-positive bacteria in solid growth media and broad spectrum activity against gram-gram-positive bacteria, gram-negative bacteria and several pathogenic fungi in liquid media Besides antimicrobial activity, Gramicidin S also displays appreciable hemolytic activity12,13

Thanatin which is an insect antimicrobial peptide from the bug podisus maculiventris has

been shown to have activity against gram-positive bacteria, gram-negative bacteria and filamentous fungi, but no hemolytic effect on porcine red blood cells14,15 Cecropins which were isolated from the hemolymph of bacteria-challenged moths and flies of the

Lepidoptera and Diptera orders are effective in killing gram-negative bacteria16-18 However, the ability to kill gram-positive bacteria and fungi varies for different cecropins5 Insect defensins are typical antimicrobial peptides which are particularly active against gram-positive bacteria but almost ineffective against gram-negative bacteria and fungi14 Some antimicrobial peptides have specificity for fungi Drosomycin

which is from the fruitfly D melanogaster preferentially kill fungi without hemolytic

activity19 A representative example of toxic antimicrobial peptide is mellitin which is a well know hemolytic peptide from the venom of honey bees20

Many antimicrobial peptides have the ability to kill malignant cells, which is attractive for clinical treatment Similar to antimicrobial activity, the difference between tumor cells and normal cells is the target of antimicrobial peptides to perform their function Charge is the main difference between tumor cells and normal cells Although the outer membrane of cancer cells is slightly negatively charged which is composed of 3-9% phosphatidylserine (PS) compared to the highly negatively charged bacterial outer membrane, some antimicrobial peptides still show higher specificity for cancer cells than for normal cells21,22 Besides PS which provides negative charges, O-glycosylated mucines which are high molecular weight glycoproteins consisting of a backbone protein

to which oligosaccharides are attached via the hydroxyl groups of serine or threonine also contribute to the negative charges22 These additional negative charges increase the interaction between antimicrobial peptides and cancer cell membranes and contribute to the selectivity of antimicrobial peptides The large number of microvilli on tumorigenic cells is another possible reason for the different susceptibilities of cancer cells and normal

cells to antimicrobial peptides23 The microvilli increase the surface area of the tumorigenic cell membranes and allow a large amount of peptide to bind to the surface 24 Several possible mechanisms have been proposed for the antitumor activity of antimicrobial peptides Two main mechanisms involve direct membrane disruption: (a) plasma membrane disruption via pore formation or micellization, (b) induction of apoptosis via mitochondrial membrane disruption22 Many antimicrobial peptides disrupt plasma membrane, leading to the death of tumor cells For example, magainins and their analogues have shown the ability to lyse hematopoietic tumor and solid tumor cells while they have little toxic effect on normal blood lymphocytes25 The induction of permeation and swelling of mitochondria which can lead to apoptosis is another direct membrane disruption way to kill tumor cells The antimicrobial peptide (KLAKLAKKLAKLAK) conjugated with a CNGRC homing domain was found to kill tumor cells by targeting mitochondria and triggering apoptosis26 Tachyplesin fused to RGD which is an integrin homing domain was shown to interact with the mitochondrial membranes of cancer cells and kill them27 Other possible mechanisms include indirect effects, for example interference with signal transduction pathways and induction of immunomodulatory effects28,29

Several antimicrobial peptides display antiviral activity in vitro with different

mechanisms Defensins which are an important family of antimicrobial peptides are remarkably antiviral against recombinant adeno-associated virus and Herpes simplex virus (HSV) with direct binding to virus to prevent envelope virus infectivity30 The antiviral activity of human neutrophil α-defensins was shown on at least two levels:

directly inactivating virus particles and affecting the ability of target CD4 cells to replicate the virus31 α-defensin-1 showed the inhibition of HIV-1 replication following viral entry and steps following reverse transcription and integration32 Gramicidin has been proved to have ability against HSV and HIV and identified to be a potent non-toxic anti-HIV agent The infection induced by many enveloped viruses including HIV and HSV leads to a drastic increase of intracellular K+, which changes the transmembrane ionic gradients and the cell membrane polarization which further affect the virus entry and viral budding However gramicidin acts as an ionophore by triggering the efflux of cytoplasmic potassium K+ from target cells, causing the depolarization of the host cell membrane Therefore, gramicidin alters the K+ balance by channel formation and adversely affects the viral infection process33-36 Dermaseptin S4, a 28-residue

antimicrobial peptide isolated from frog skin, shows strong antiviral activity in vitro

against HSV-1 at micromolar doses, probably through interference with virus-cell surface interactions37 The interaction between dermaseptin S4 and HIV-1 indicate that the activity is directed against HIV-1 particles by disrupting the virion integrity38 The cathelicidin family of antimicrobial peptides also has antiviral activity Indolicidin is shown active against HIV-1 and the activity is temperature sensitive This indicates a membrane-mediated antiviral mechanism which is a direct interaction of peptide with the virus envelope, leading to permeation of the envelope and eventually lysis of the viral particles39 Human cathelicidin (LL-37) exhibits rapid and efficient killing of HSV-1 and suggests a most likely mechanism of permeabilization of the external lipid membrane However, the antiviral activity of LL-37 against vaccinia virus is attributed to direct disruptive action on the viral envelope40 Human LL-37 and porcine Protegrin-1 are

found to specifically inhibit lentiviral and retroviral vectors, which suggests that they directly interact with the vector particles41 Except these, other antimicrobial peptides which have different antiviral activity include lactoferrin and its small peptide fragment from its N-terminal domain, lactoferricin, caerin1.1, caerin1.9, maculatin1.1, cecropin B and its analogues, cecropin A, melittin, magainin 1 and 2, and hybrid peptides42-46

2.2 Classification of antimicrobial peptides

Until now, more than 800 antimicrobial peptides have been identified in eukaryotes (http://www.bbcm.units.it/~tossi/amsdb.html , Antimicrobial Sequences Database) They are widely distributed in invertebrates, amphibians, plants, mammals and humans5 The primary structure of these antimicrobial peptides is so diverse that it is difficult to find the same peptide sequence from two different species, even when they are closely related The diversity of the antimicrobial peptides in the amino acid sequence in different species indicates the adaptation of the different species to the environment When a species suffers a particular microbial infection, single mutations will greatly change the biological activity of individual peptides Those mutational peptides which do not assure host survival will not be retained Although the individuals might suffer, the species could survive since the beneficial antimicrobial peptide will be selected47,48 Because there is not much similarity in their primary structures, it is common to classify the antimicrobial peptides based on their secondary structures Generally the antimicrobial peptides can be divided into four classes: α-helices, β-sheet, extended structures and looped structures48-51 The representative antimicrobial peptides of each class are presented in Table 2.1

Table 2.1 Representative antimicrobial peptides with different secondary structures1,4,47,49,51,52

α-helix

Cecropin A KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK Silk moth Hyalophora cecropia

Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGPR Porcine small intestines

laevis)

laevis)

LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Human leukocytes, epithelia

β-sheet

HNP-1 (α-defensin) ACYCRIPACIAGERRYGTCIYQGRLWAFCC Human (Homo sapiens)

HBD-2 (β-defensin) MRVLYLLFSFLFIFLMPLPGVFGGIGDPVTCLKSGAICHPV

FCPRRYKQIGTCGLPGTKCCKKP

Human (Homo sapiens)

horseshoe)

Extended

Bactenecin 5 RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP Cow (Bos taurus)

Histatin I DSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN Human (Homo sapiens)

Diptericin DEKPKLILPTPAPPNLPQLVGGGGGNRKDGFGVSVDAHQ

KVW TSDNGRHSIGVTPGYSQHLGGPYGNSRPDYRIGAGYSYN

F

Black blowfly (Phormia terranovae)

Looped structure

an antimicrobial peptide has effect on its antimicrobial activity54-56

β-sheet conformation

Compared to the linear α-helical peptides, β-sheet peptides are cyclic peptides composed

of two or more β-strands which are stabilized by intramolecular two or more disulfide bonds They exist in aqueous solution in β-sheet conformation and are further stabilized upon interaction with membranes57 The typical examples of this class are defensins, polyphemusins, tachycitins and drosomycin1 Defensins or defensin-like peptides are widely distributed in insects, mammals and humans These peptides can be divided into

several sub-classes, such as α-defensin, β-defensin and θ-defensins14,58-60 θ-defensins were discovered recently in rhesus macaque neutrophils61 α-defensins contain from 29 to

35 amino acid residues and three disulfide bonds which stabilize the three-strands β-sheet These disulfides bonds form cysteine pairing: Cys1-Cys6, Cys2-Cys4 and Cys3-Cys5 β-defensins contains from 34 to 47 amino acids and are linked differently from the α-defensins: Cys1-Cys5, Cys2-Cys4 and Cys3-Cys61 Most of the insect defensins are composed of 36 to 46 amino acid residues and all insect defensins have the same cysteine pairing: Cys1-Cys4, Cys2-Cys5 and Cys3-Cys614 Polyphemusins and tachyplesins are β-

sheet antimicrobial peptides from American horseshoe crab Limulus polyphemus and Japanese horseshoe crab Tachypleus tridentatus, respectively62 They are 17-18 amino acid residues in length, containing two disulfide bonds and have an amidated C-terminal arginine4 Drosomycin which consists of 44 amino acid resides is a cysteine-rich antifungal peptide, including 4 internal disulfide bonds: Cys1-Cys8, Cys2-Cys4, Cys3-Cys6 and Cys4-Cys719,63

Looped peptides

This class of antimicrobial peptides forms a hairpin-like β-turn structure, usually consisting of one disulfide bond or backbone covalently cyclized The typical example of this class is bactenecin or dodecapeptide from cattle neutrophils Bactenecin is composed

of 12 amino acid residues including 2 cysteines and is shown to form a β-turn structure regardless of its environment64 This peptide has weak ability against gram-negative bacteria Reduction of the disulfide bond or replacing cysteine with serine will cause the peptide to lose the activity against gram-negative bacteria The interaction of the

linearized peptides with cell membrane is different from that of cyclic peptide with lower activity for outer membrane but effective in permeabilizing the cytoplasmic membrane The linearized peptides are unstructured in aqueous solution, but adopt β-turn structure upon interaction with membranes64 Those peptides whose backbones are covalently cyclized include gramicidin S and polymyxin B48

Linear extended peptides

The class of linear extended peptides which lack a defined secondary structure is rich in one or more specific amino acids The members of this class include histatin, PR-39, tritrpticin, indolicidin and some glycine-rich peptides1 Histatin which is produced by human salivary glands has a high proportion of His residues (18-29%) They are highly cationic with 7 to 8 positive charges and have antifungal activity65 PR-39 which is found

in porcine neutrophils is an antimicrobial peptide rich in proline and arginine This peptide has antimicrobial activity and is able to translocate model membranes6 Tryptophan is not a common abundant residue in peptide sequences However tryptophan

is rich in tritrpticin and indolicidin with 3 or 5 of their 13 amino acid position, respectively66,67 Tryptophan is of particular interest because the position of this residue can indicate the location of itself near the membrane and water interface when the peptide interacts with a membrane68,69 It has shown that these two peptides are active against both gram-positive and gram negative bacteria1 The tertiary structure has been solved to show that a boat-like extended structure formed upon interaction with micelles The region which is rich in tryptophan in the middle of the peptides interacts with one layer of the membrane and the two termini are oriented toward the aqueous environment70-72

Indolicidin which permeabilizes bacteria is able to form conductance channels on the membrane, which suggests self-association73 Glycine-rich peptides can be identified in both amphibians and insects A number of antimicrobial peptides from insects which are proline-rich or glycine-rich may be modified by the addition of glycosyl groups74,75

2.3 Structural features of antimicrobial peptides

Antimicrobial peptides have broad spectrum ability to defend against microorganisms regardless of the classification Most of the peptides are cationic and have amphipathic structures, which are significant factors for determining the biological activity of antimicrobial peptides Besides these two parameters, their activity also depends on other factors, for example peptide size, helicity, hydrophobicity and disulfide bonds5,54 It is difficult to determine the individual importance of each factor because they influence each other and are difficult to separate However, some information regarding factors related to the biological activity of antimicrobial peptides still can be obtained The study

on the structural features of antimicrobial peptides is important to enhance the understanding of antimicrobial peptides and provide useful information on rational peptide design

Charge: Most of antimicrobial peptides are cationic The net charge usually ranges from

+2 to +9 and varies with pH76 The positive charge which facilitates the binding of antimicrobial peptides to negatively charged membranes is an important reason for selectivity of antimicrobial peptides Even in some net negatively charged antimicrobial peptides, a local cationicity is needed to interact with lipid head groups Subtilosin A is

an anionic antimicrobial peptide with net charge (-2), however the lysine which is located

at one terminus facilitates the interaction of two termini of subtilosin A with membranes77 With increasing net positive charge, both antibacterial and hemolytic activity increased for paradaxin5 High positive charge increases the electrostatic interaction between peptides and lipids However, the charge cannot be increased too high, because an excessive charge can have a deleterious effect on activity78 The strong electrostatic interactions may induce the repulsion between the positively charged side chains and obstruct pore formation The high charges may also change the structure of peptides, which influences the peptide activity79 For example, magainin analogues have been shown to form smaller, less stable, short lived pores and faster translocation of peptides across the membrane with increased charges5 Studies have shown that +4 to +6

is an optimal charge range for antimicrobial peptides to interact with membranes5,80

Amphipathicity: A large number of studies have shown that amphipathicity is a critical

factor of biological activity of antimicrobial peptides in spite of the secondary structures78,81-84 This parameter measures the spatial separation between hydrophilic and hydrophobic side chains It is generally determined by calculating the hydrophobic moment, the vector sum of the hydrophobicities of each amino acid residue The mean hydrophobic moment per residue is used to compare amphipathicity of peptides with different length55,56,78,85-87 It has been shown that amphipathicity affects the antimicrobial activity and hemolytic activity On one hand increased amphipathicity leads to increased hemolytic activity82 On the other hand the effect of amphipathicity on antimicrobial activity is determined by the peptide studied Antimicrobial activity decreases when the

amphipathicity is lower than a certain threshold value However, above this threshold value, increased amphipathicty may also cause decreased antimicrobial activity78 The study of 14-residue analogues of Gramicidin S indicated that increased amphipathicity results in decreased antimicrobial activity82

Hydrophobicity: Hydrophobicity is defined as the average of the numeric hydrophobicity

values of all residues of a peptide86, representing the ability of antimicrobial peptides to move from an aqueous phase into a hydrophobic phase78 The relationship between antimicrobial activity and hydrophobicity is still controversial However the dependence

of hemolytic activity on the hydrophobicity is much clearer than antimicrobial activity78 Most of studies have shown that an increased hydrophobicity is related to an enhanced permeabilizing activity on zwitterionic lipids, which decreases the selectivity of peptides for bacterial membranes The replacement of leucine by lysine which decreases the hydrophobicity caused considerably decrease in hemolytic activity whereas the inverse substitution enhanced the hemolytic activity, indicating a direct relation existing between hydrophobicity and hemolytic activity88 Magainin 2 amide and its designed analogues with varied hydrophobicity and largely unchanged other structure parameters showed the strong dependence of hemolytic activity on hydrophobicity89 In addition, the effect of hydrophobicity on the biological activity of antimicrobial peptides is also represented by the hydrophobic/hydrophilic angles78

Helicity: Helicity is an important factor influencing the biological activity of α-helix

antimicrobial peptides Those antimicrobial peptides with high helicity have a higher

potential to obtain better spatial distribution of polar and non-polar amino acids, resulting

in high amphaphilicity Thus an increase in helicity might lead to an increase in biological activity of antimicrobial peptides The studies of appropriate amino acid substitutions which increase the helicity of antimicrobial peptide showed an enhancing membrane activity90-93 However the substitution of amino acids not only modifies the helicity, but also changes the other parameters including hydrophobicity and charges, thus the relation between helicity and biological activity is limited Replacement of L-amino acids by corresponding enantiomer D-amino acids disturbs the local helicity while keeping other parameters constant, such as hydrophobicity and charges, which might provide more information concerning the role of helicity in the antimicrobial activity and hemolitic activity78 A substitution of a series of designed antimicrobial peptides based

on a cationic amphipathic KLAL model showed that an increase in helicity led to an increase in biological activity, especially the permeabilizing activity against neutral lipid vesicles 94 Thus high helicity is favorable toward hemolytic activity However, the amphipathicity, hydrophobicity and helicity are well correlated and a change in one parameter will change other parameters with high possibility

Disulfide bonds: Most of antimicrobial peptides with β-sheet or β-turn structures have

one or more disulfide bonds These disulfide bonds do not have a unique function depending on the peptide and activity assayed In some instances, a reduction of disulfide bonds or replacement with other amino acids has little effect on activity However, in other cases the activity can be completely changed51,95,96 For example, reduction causes insect defensin to lose channel-forming activity, but have not much effect on mammalian

defensins97 The reduction of disulfide bonds not only changes the activity of antimicrobial peptides, but also changes the structure and mechanism Reduction of both rabbit and human defensins leads to changes in the mechanism from all-or-none to gradual leakage98 Tachyplesins also change their mechanism from channel formation to

a detergent-like mechanism upon reduction99,100 In addition, intermolecular disulfide bonds also play a role in the activity of antimicrobial peptides The one cysteine of the S3 peptide which is derived from the Sushi 3 domain of Factor C allows the formation of S3 dimers The S3 dimer is capable of disrupting the LPS micelles whereas the reduction of the dimer into monomer makes S3 lose this ability101

L- and D- amino acid composition: The biological activity of antimicrobial peptides is

also related to the L- or D- enantiomer However the effect of enantiomer of the amino acid is dependent on whether the replacement affects the structure of the peptide and the amphipathicity D-melittin, D-magainin and D-cecropin derivatives which are all-D enaniomers are found to be a mirror image of the corresponding L isomers using circular dichroism However the D isomers have the same biological activities as those of the native peptides102 The action of antimicrobial peptides on the membrane indicates that the mechanism of antimicrobial peptides does not involve a stereoselective interaction with a chiral enzyme or lipid or protein receptor102 However if some amino acids of a peptide is replaced by D-enantiomer, which changes the conformation of the peptide, the biological activity might be greatly influenced The single enantiomeric substitution of the amino acid in an analogue of Gramicidin S shows a disrupted β-sheet structure and different antimicrobial activity and hemolytic activity82

2.4 Biological membranes

A variety of studies have shown that the target of antimicrobial peptides is the cell membrane, which provides protection for microbes and cells The attractive property of antimicrobial peptides is the selectivity for killing microbes without harming mammalian cells This selectivity is attributed to the different components of the bacterial and mammalian cell membranes It has been shown that many antimicrobial peptides have higher affinity for bacterial membranes than for mammalian cell membranes due to electrostatic interactions

Bacterial membranes have a unique composition and structure which are different from mammalian cell membranes as shown in Fig 2.1103 Out of the cytoplasmic membrane there is an outer membrane and a thinner peptidoglycan layer in gram-negative bacteria and a thicker peptidoglycan layer in gram-positive bacteria There is a large amount of lipopolysaccharide (LPS) on the outer leaflet of outer membrane of gram-negative bacteria and teichoic acids on the peptidoglycan layer of gram-positive bacteria, both of which are negatively charged In addition, the cytoplasmic membrane of bacteria also consists of a high percentage of negatively charged phospholipids Compared to the outer leaflet of the mammalian cell membrane which is predominantly composed of zwitterionic phosphatidylcholine and sphingomyelin phospholipids, bacterial membranes are highly negatively charged which facilitate the interaction with cationic antimicrobial peptides Some tumor cells lose the lipid asymmetry and have a more anionic character

on the outer leaflet of their plasma membrane, which facilitates the binding to cationic antimicrobial peptides104

Fig 2.1 Schematic drawing of gram-positive and gram-negative bacterial membranes

LPS, also known as endotoxin, which is a major component of the outer membrane in gram-negative bacteria, activates the host defense system of most organisms, playing an important role in the pathophysiology105 The release of LPS in host could stimulate enhanced secretion of various pro-inflammatory cytokines such as tumor necrosis factor-

α (TNF-α) and interleukin-6 (IL-6) Although pro-inflammatory cytokine secretion is essential for inducing local inflammatory response, overproduction of these cytokines may lead to severe septic shock, organ failure and even death after gram-negative bacterial sepsis106-108 LPS which is negatively charged is composed of three parts: O-antigen, polysaccharide and lipid A105 Lipid A is the conserved bioactive component of

hydrocarbon chains and a β-D-GlcN-(1-6)-α-D-GlcN disaccharide with two phosphoryl groups The lipid A moiety is the most conserved part between LPS of different serotypes109 It has been shown that lipid A is the main interacting partner with antimicrobial peptides110 The polysaccharide is the linker between lipid A and O-antigen and is constituted by two cores The inner core is mainly composed of 3-deoxy-d-manno-octulosonic acid (Kdo) and l-glycero-d-manno heptose (Hep), and the outer core includes hexoses and hexosamines111 Lipid A and Kdo are prerequisite for forming a minimum LPS molecule112 O-antigen which is an important surface antigen extends into the environment109,113 It consists of 0–50 repeating subunits which are composed of 1–8 glycosyl residues111 For different bacterial serotypes, there are great differences in the length and composition of the polysaccharide and O-antigen

Using antimicrobial peptides to neutralize LPS, especially lipid A, is believed to be an optionally effective approach to kill bacteria LPS serve as the first barrier for antimicrobial peptides interacting with bacterial membrane The interaction of antimicrobial peptides with LPS is believed to use a process named self-promoted uptake48,114,115 Cationic antimicrobial peptides first interact with divalent cation binding sites on surface LPS Due to the much higher affinity of antimicrobial peptides for LPS, the native divalent cations such as Ca2+ or Mg2+, which are used to stabilize LPS, are displaced and the outer membrane is distorted This results in the loss of the barrier property of the outer membrane and permits passage of a variety of molecules including antimicrobial peptides themselves across the outer membrane to contact with cytoplasmic membrane The LPS neutralization of antimicrobial peptides and promotion of the uptake

of other agents show synergy with conventional antibiotics especially against antibiotic resistant mutants116

The binding affinity for LPS of antimicrobial peptides is not directly related to the antimicrobial activity Studies of Gramicidin S analogues have shown that the high interaction between antimicrobial peptides with LPS may lead to decreased ability to penetrate into the cytoplasmic membrane because of the large accumulation at the outer membrane82 Additionally the antimicrobial peptides which bind to the bacterial surface

as oligmers are difficult to diffuse through the LPS layer into the target membrane and show low antimicrobial activity Therefore the disruption of the outer membrane is not a lethal factor for bacteria The antimicrobial peptides pass through the outer membrane, reach and interact with the cytoplasmic membrane, either forming pores or disrupting the membrane, and eventually lead to bacterial death50

The cytoplasmic membrane is crucial for biological functions The major component phospholipids are especially important because they form the backbone of the membrane Antimicrobial peptides perform their function through non-specific mechanisms and the phospholipids are their essential interacting target Cationic antimicrobial peptides preferentially bind to the membranes containing acidic phospholipids such as PG or PS due to the strong electrostatic interactions117-119 Many peptides which have shown higher affinity for negatively charged lipids have higher potency to kill bacteria Besides the charge of lipids, there are also other factors which influence the peptide-lipid interaction, such as fluidity of membrane, flexibility of membrane and membrane curvature117 Most