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Characterization of bactenecin : a small antimicrobial cationic peptide Wu, Manhong 1999

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Characterization of Bactenecin: A Small Antimicrobial Cationic Peptide By Manhong W u B. Sc. Fudan University, Shanghai, 1988 M . Sc. University of British Columbia, 1993  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY  in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming tdvthe required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A September, 1998 © M a n h o n g W u , 1998  In  presenting  degree freely  at  the  available  copying  of  department publication  this  of  in  University  of  for reference  this or  thesis  thesis by  this  for  his thesis  or  partial  and study. scholarly her  the  requirements  I further agree that  purposes  may  representatives.  be  It  for financial gain shall not  Department The University of British C o l u m b i a Vancouver, Canada  (2788)  of  British Columbia, I agree that  permission.  DE-6  fulfilment  is  the  an  permission for  granted allowed  advanced  Library shall make by  understood be  for  the that  without  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Bactenecin is a 12-amino acid-long cyclic peptide from bovine neutrophils. It has the sequence: R L C R I V V I R V C R , and was cyclized by formation of a single disulphide bond. Computer modeling suggested it was amphipathic, with a central hydrophobic ring and positive charges located at both the C - and N-termini.  Circular dichroism (CD)  spectral studies were consistent with the computer model, in showing that bactenecin is rigid, (3-turn structure regardless of its environment. C D spectral studies showed that two linear variants of bactenecin were more flexible, then adopted random structure in phosphate buffer, a-helical structure in trifluoroethanol and (3-sheet structure in the presence of liposomes. Bactenecin was shown to have moderate antimicrobial activity against three wildtype Gram-negative bacteria, but was inactive against several Gram-positive bacteria. Disruption of the disulphide bond abolished the antimicrobial activity against the wild type  Gram-negative bacteria, but had in improved antimicrobial activity  Staphylococcus epidermis  and  Entercoccus faecalis.  Both the native cyclic and linear  bactenecins interacted with the outer and cytoplasmic membranes of differently.  against  Escherichia coli  Nature bactenecin bound and permeabilized the outer membrane better than  the linear variants, which explains its better activity against the wild-type Gram-negative bacteria. membrane.  However, bactenecin  had poor activity in depolarizing the  cytoplasmic  Conversely the linear variants demonstrated poor interaction with the outer  membrane, yet were effective in depolarizing the cytoplasmic membrane.  These results  ii  suggest that cyclization was  important  for bactenecin  to  interact  with the  outer  membrane, and furthermore, that cyclic bactenecin could act by a fundamentally different mechanisms from its linear variants. Bactenecin was proposed to have a mechanism of action which involves other cellular targets than the bacterial membrane. Structure:function studies implied that increasing the number of positive charges, by introduction of additional arginine residues at both the N - and C-termini, resulted in improved activity.  Linear variants were more active against Gram-positive bacteria.  Introduction of the hydrophobic residue, tryptophan, into the loop of cyclic bactenecin improved the spectrum.  antimicrobial activity  and dramatically broadened  the  antimicrobial  Two interesting candidates as potential therapeutic agents were identified,  B a c 2 A - C N and Bac2R,W. These two peptides had good antimicrobial activity against a wide range of bacteria, including some clinically significant pathogens, and low toxicity.  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENT  xi  INTRODUCTION  1  A. B. C. D.  Antimicrobial Polycationic Peptides Loop-Structured Cationic Peptides and Bactenecin Therapeutic Potential Aims of This Study  2 12 16 17  MATERIALS AND METHODS A. Bacterial Strains and Growth Conditions B. Chemicals C. Recombinant Expression of Peptides D. Purification of the Recombinant Peptides E. Peptide Preparation F. Structural Studies G. Antimicrobial Activity and Hemolytic Activity H. Membrane Permeabilization Assays  18 18 18 21 25 26 30 31 32  RESULTS  35  CHAPTER I: RECOMBINANT EXPRESSION OF CATIONIC PEPTIDES A. Introduction B. Recombinant Expression of Three Small Cationic Peptides in S. aureus C. Production of Bactenecin in E. coli D. Summary  35 35 36 46 49  CHAPTER TWO: ANTIBACTERIAL MECHANISM OF BACTENECIN A. Introduction B. Antimicrobial Activity C. Secondary Structure by Circular Dichroism D. Interaction with Bacterial cells E. Summary  50 50 52 54 54 62  iv  CHAPTER THREE: INNER MEMBRANE PERMEABILITY 64 A. Introduction 64 B. Fluorescence Quenching of diSC3-(5) by E. coli DC2 cells 66 C. The relationship of K concentration outside the cells and the fluorescence intensity in the presence of valinomycin 66 D. Antimicrobial activities in the presence of potassium chloride 72 E. The interaction of adielical cationic peptides with the cytoplasmic membrane... 72 F. The interaction of P-structured cationic peptides with the cytoplasmic membrane 75 G. The interaction of extended structured cationic peptides with the cytoplasmic membrane 78 H . The interaction of looped structured cationic peptides with the cytoplasmic membrane 78 G. Summary 84 +  CHAPTER FOUR: STRUCTURE AND FUNCTION RELATIONSHIPS A. Introduction B. Linearization C. Positive Charges E. Disulphide bond  85 85 86 90 98  DISCUSSION A. Overview B. Recombinant Expression of Cationic Peptides C. Antimicrobial Mechanism of Bactenecin D. Design of Novel Peptides with Improved Activity E. Potential Application of Bactenecin F. Future Studies  102 102 103 107 113 114 115  REFERENCES  120  LIST O F T A B L E S Table I: Loop-Structured Cationic Peptides  12  Table II: Bacterial Strains  19  Table III: Amino A c i d Sequences of Bactenecin and its Derivatives  28  Table IV: Amino A c i d Sequences of other Cationic Peptides Used in This Study  29  Table V : M I C s of Three Partially Purified Cationic Peptides Expressed in S. aureus  46  Table VI: M I C s of Bactenecin, its Linear Variant Bac 2S and its Reduced Form Lin-Bac 53 Table VII: M I C s of E.coli D C 2 in the Presence and Absence of K  73  +  Table VIII: M I C s of Linear, Amidated Bactenecin Derivatives  88  Table IX: M I C s of Bactenecin Derivatives in the Reduced F o r m  89  Table X : M I C s of Positive-Charge Bactenecin Derivatives  94  Table X I : M I C s of Hydrophobicity Bactenecin Derivatives  97  Table XII: Agglutination Activities of Bactenecin and its Derivatives on Human Red Blood Cells  100  Table XIII: Influence o f Bactenecin Structural Modifications on Antimicrobial Activity 101  vi  LIST O F FIGURES Figure 1: A m i n o acid sequences of the three recombinantly expressed cationic peptides. 23 Figure 2: Sequences o f oligonucleotides  24  Figure 3: Production o f protein A/cationic peptide fusion proteins in S. aureus  37  Figure 4: Cleavage of cationic peptides from protein A  39  Figure 5A: Elution profile of the CNBr-digested protein A/apidaecin fusion from a Biogel PI00 Column Figure 5B: Purification o f apidaecin on a Biogel P100 column  40 41  Figure 6A: Elution profile of the CNBr-digested protein A/bactenecin fusion from a Biogel P100 Column. Figure 6B: Purification of bactenecin on a Biogel PI 00 Column  42 43  Figure 7A: Elution profile o f the CNBr-digested protein A/indolicidin fusion from a Biogel PI00 Column  44  Figure 7B: Purification of indolicidin on a Biogel PI00 Column  45  Figure 8: Production o f bactenecin in E. coli  48  Figure 9: A m i n o acid sequence o f bactenecin and its linear variants  51  Figure 10A: C D spectra o f bactenecin, its reduced form and bac2S in phosphate buffer. 55 Figure 10B: C D spectra of bactenecin, its reduced form and bac2S in the presence of P O P C / P O P G liposomes  56  Figure 10C: C D spectra of bactenecin, its reduced form and bac2S in 60% (v/v) T F E . . .57 Figure 10D: C D spectra o f bactenecin, its reduced form and bac2S in l O m M S D S  58  Figure 11: Binding o f peptides to L P S as assessed by their ability to displace dansyl polymyxin B from E. coli UB1005 L P S  60  Figure 12: Pep tide-induced outer membrane permeabilization measured by the N P N uptake assay in E. coli U B 1005 Figure 13: Fluorescence quenching of diSC3-(5) by log-phase E. coli cells  61 67  Vll  Figure 14: Effect of external KC1 concentration on the fluorescence intensity of 0.4 p M diSC3-(5) incubated with E. coli D C 2 cells and valinomycin  68  Figure 15: Kinetics of fluorescence intensity changes of diSC3-(5) incubated with E. coli DC2  cells in the presence of valinomycin and KC1  70  Figure 16: The effect of 0.1 M KC1 on the dissipation of cytoplasmic membrane potential caused by gramicidin S  71  Figure 17: Kinetics o f fluorescence intensity changes of diSC3-(5) incubated with E. coli DC2  in the presence of a-helical cationic peptides  74  Figure 18: Effect of a-helical cationic peptides on the fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 cells  76  Figure 19: Kinetics of the fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 in the presence of p-structured cationic peptides  77  Figure 20: Effect of B-structured cationic peptides on fluorescence intensity of diSC3-(5) incubated with E. coli D C 2 cells  79  Figure 21: Kinetics o f fluorescence intensity changes of diSC3-(5) incubated with E. coli DC2  in the presence of extended cationic peptides  80  Figure 22: Effect of extended cationic peptides on the fluorescence intensity of diSC3-(5) incubated with E. coli D C 2 cells  81  Figure 23: Kinetics of fluorescence intensity changes of diSC3-(5) incubated with E. coli DC2  in the presence of loop-structured cationic peptides  82  Figure 24: Effect of loop-structured cationic peptides on the fluorescence intensity of diSC3-(5) incubated with E. coli D C 2 cells  83  Figure 25: Computer generated model of bactenecin  91  Figure 26: Computer generated model of bacR,P  92  Figure 27: Computer generated model of bacP  117  Figure 28: Computer generated model of bacW  118  Figure 29: Computer generated model of bac2R,W  119  viii  LIST O F ABBREVIATIONS A U : Acid-Urea C D : Circular Dichroism C E M E : cecropin/melittin hybrid peptide (CA1-8M1-18) C F U : colony forming unit C N B r : cyanogen bromide diS-C3-(5): 3, 3-dipropylthiacarbocyanine E D T A : ethylenediamine tetraacetic acid F P L C : fast protein liquid chromatography H E P E S : (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) sodium salt H N P : human neutrophil peptide I P T G : isopropyl-P-D-thiogalactoside L B N S : Luria Broth Normal Salt L P S : Polyliposaccaride M I C : M i n i m u m Inhibitory Concentration N M R : nuclear magnetic resonance N P N : 1-N-phenylnaphtylamine O D : optical density P A G E : polyacrylamide gel electrophoresis P C R : polymerase chain reaction P O P C : 1 -pamitoyl-2-oleoyl-sn-glycero-3 -phosphocholine POPG:  l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol  ix  N A P S : Nucleic Acid/Protein Service unit RP: reverse phase rpm: revolutions per minute SDS: sodium dodecyl sulfate T F A : trifluroacetic acid T F E : trifluroethanol U V : ultraviolet  ACKNOWLEDGEMENT I would like to thank my supervisor Bob Hancock, for his excellent supervising, his patience, and his constant encouragement.  I would like to thank the members in  Hancock's lab, present and past who were always helpful.  In addition, I would like to  thank the members of my committee, Drs Tony Warren, Brett Finlay, B i l l M o h n for their helpful discussion and advice.  Finally I would like to thank my parents, my brother  Haiying W u and my friends (especially Liping, Liyue, Kewen and Hua) who were always there for me and encouraged me to carry on.  The financial support of the British  Columbia Science Council and of Micrologix Biotech Inc. was also greatly appreciated.  XI  INTRODUCTION  The rapid emergence of antibiotic resistance has been of great concern in recent years. Some bacteria have developed resistance to virtually all antibiotics available in the world due to extensive clinical usage o f antibiotics (Neu, 1992).  Resistance i n the  Enterococci and Staphylococci is becoming particularly troublesome (McManus, 1997). Efforts have been made to prevent and control the resistance problem, including public health efforts to reduce unnecessary usage of antibiotics in humans and animals, as well as the development of new antimicrobial agents (Cohen, 1992). The latter has been the focus o f research scientists interested in this area. Based on our current understanding of the molecular mechanisms o f bacterial resistance, researchers continue to improve the existing antimicrobial agents by targeting "resistance factors" (Setti and Micetich, 1998). It seems that bacteria can easily develop resistance by the same mechanisms to the new analogs of existing antibiotics. Therefore, the development of a new class o f antibiotics has become increasingly important. A m o n g the possible candidates, a group of antimicrobial cationic peptides has attracted increasing research and clinical interest due to their unique properties.  This  group o f gene-encoded "endogenous peptide antibiotics" (Martin et al., 1995) is widely distributed in nature, and are key components of the non-specific defense systems for plants and animals (Boman, 1995). For conventional antibiotics to reach their targets and kill microorganisms, they have to overcome two major diffusion barriers, the outer and cytoplasmic membranes. Bacteria can easily develop mechanisms to reduce accessibility to these barriers. Cationic peptides have been proposed to exert their antimicrobial  1  activity by perturbing the barrier function of bacterial membranes.  Conventional  antibiotics function by an entirely different mechanism, in that they usually interfere with either cell wall or macromolecular synthesis (Neu, 1992).  Cationic peptides have thus  become a new source of potentially novel antimicrobial agents.  Therefore, a detailed  characterization and understanding of their structures and mode of action, and a correlation of structure with function is essential in assessment of their therapeutic prospects.  A. Antimicrobial Polycationic Peptides  1. In Nature Antimicrobial polycationic peptides have been found in a variety of sources, from prokaryotes (bacteria) to eukaryotes (plants and animals, such as amphibians, mammals, insects) (Hancock et al, 1995). are:  Some examples of the best known and studied peptides  1) from mammals, classic defensins (Kagan et al., 1994); 2) from amphibians,  magainins from the granular glands present in the skin of Xenopus laevis (Duchohier, 1994); 3) from insects, insect defensins (Lehrer, 1993), cecropins from the North American silk moth Hyalophora  cecropia (Hultmark et al., 1993), and melittins from the  venom of the honey bee Apis mellifera  (Dempsey,  1990); 4) from plants, thionins  (Bohlmann, 1994); and 5) nisin from bacteria Lactococcus lactis (Jung and Sahl, 1991). In recent years, it has become clear that these endogenous peptide antibiotics constitute part of the first line of host defense (Boman, 1991). Cationic peptides can be induced and synthesized much more rapidly than immuoglobulins upon infection, before  2  the adaptive immune system is activated, and they function without the high specificity and memory of immunoglobulins or immune cells (Boman, 1995; Nissen-Meyer and Nes, 1997).  In mammals, antimicrobial peptides  are present at high concentrations  in  phagocytes (e.g. macrophages, neutrophils, N K cells) and mucosal epithelial cells (e.g. Paneth cells).  Human defensins constitute about 5-7% of total cellular protein in  neutrophils (Ganz, 1987) and participate in the early phases of defense against invading microbes by the oxygen-independent pathway of neutrophils, as an adjunct to the oxygendependent pathway (Lehrer, 1991). Besides being antimicrobial, defensins may also play a role in inflammation, tissue injury and repair (Lehrer, 1993). In lower life forms, such as invertebrates, which have no adaptive immunity, cationic peptides  are the major defensive system against infection (Boman, 1995).  Insects produce cationic peptides in their fat bodies and haemolymph, where they are induced upon bacteria challenge (Boman, 1995; Hoffmann and Hetru, 1992). Frog skin contains  abundant antimicrobial peptides  contaminated pond water (Zasloff, 1987).  to keep wounds  safe from infections  in  Cationic peptides also function to keep the  natural flora o f bacteria in a steady state, in a variety of different niches such as the skin, the mouth, and the intestine (Boman, 1995). The natural cationic peptides of animals and plants are synthesized ribosomally as precursor and are processed into their mature forms by cleavage of a signal peptide and a prosequence (Zanetti, 1995; Hancock, 1997).  3  2. In Vitro Activity The above-mentioned cationic peptides and many others exhibit potent activities against a rather broad spectrum of microorganisms including bacteria, enveloped viruses, fungi and tumor cells.  For example, defensins are active against Gram-positive and  Gram-negative bacteria (including spirochetes and mycobacteria), fungi and enveloped viruses (Lehrer et al., 1993). Magainins (Bevin and Zasloff, 1990) also exhibit antifungal and  antiviral activities  in  addition  to  their  anti-bacterial  activities.  Defensins  (Lichtenstein, et al., 1988) and magainin (Cruciani, et al., 1991) have also been shown to be active against tumor cells. Most interestingly, these cationic peptides (e.g. tachyplesin I) not only inhibit the growth of normally susceptible Gram-positive and negative bacteria but also inhibit with equal potency clinically important antibiotic-resistant strains e.g. methicillin-resistant S. aureus (Ohta et al., 1992). antimicrobial activity varies.  However, the spectrum of  For example, classical defensins (Kagan, et al., 1994) and  insect defensins (Hoffmann and Hetru, 1992) are more active against Gram-positive than Gram-negative bacteria. In contrast, cecropins (Boman, 1991) and magainins (Bevin and Zasloff, 1990)  exhibited greater potency against Gram-negative bacteria than Gram-  positive bacteria. The reasons for this selectivity against Gram-positive or Gram-negative bacteria have not been made clear in the literature to date.  3. Structural Features and Categories Polycationic peptides show significant diversity in their sizes, sequences and structures. compositions  They range from 12 to 46 amino acids long with diverse amino acid (Hancock, 1997).  Despite their diversity, antimicrobial peptides share  4  common structural features, being virtually all cationic and very often amphiphilic. The cationic antimicrobial peptides have a net charge of at least +2 at neutral p H , usually due to the presence of arginine or lysine residues in their amino acid sequence (Hancock, 1997). Their secondary structures often contain a hydrophobic domain and a hydrophilic domain. The basicity and amphipathicity of cationic peptides are essential for their antimicrobial activities (Saberwal and Nagaraj, 1994; Hancock, 1997). The hydrophilic (positively charged) surface facilitates the interaction of the peptides with the negatively charged bacterial surface, e.g. L P S on the outer membrane of Gram-negative bacteria, teichoic acid on the Gram-positive bacteria, or negatively charged headgroups of the phospholipids in the lipid bilayer (Wade et al., 1992; Piers et al., 1994; Kagan et al., 1994).  Some evidence showed that the property of amphipathicity was important to  permit the antimicrobial peptides to interact with the membrane and to form channels or pores (Saberwal and Nagaraj, 1994; Maloy and Kari, 1995). Based on their known or assumed secondary structures, polycationic peptides could be categorized into four major groups: 1) P-sheet structures stabilized by two or three disulphide-bridges; 2) amphipathic a-helices often comprising a helix-turn-helix arrangement; 3) extended polyprohelices with a predominance of one or more amino acids like proline, or tryptophan; and 4) loop-structures  containing only a single  disulphide-bridge. These are described immediately below, except for the loop-structured peptides which are described in section 5.  5  3.1 P-structured peptides. Cysteine-rich cationic peptides usually contain p-strands stabilized by two or three disulphide-bridges. Antimicrobial peptides with six cysteine residues generally have some sequence conservation and have been classified as defensins (Ganz and Lehrer, 1994). There are three subgroups of defensins, classical defensins, P-defensins and insect defensins.  They differ from each other in the spacing and connectivity o f their six  cysteine residues. Classical defensins contain triple-stranded antiparallel P-sheet without the presence o f a-helical stretches (Zhang et al., 1992; Pardi et al., 1992).  In contrast,  insect defensins consist o f a short a-helix and an antiparallel P-sheet connected by a loop (Hoffmann and Hetru, 1990). The three dimensional structure of P-defensins is unknown at present. The details o f the secondary and tertiary structures of defensins are known from two-dimensional nuclear magnetic resonance, circular dichroism, Fourier transform infra red spectroscopy, and X-crystallography (Bach et al., 1987; Pardi et al., 1988; Selsted and Harwig, 1989; H i l l et al., 1991; Zhang, et al., 1992;). Human defensin 3 has been shown by X-ray crystallography to be present as basket-like dimer with the top half containing the polar residues and the lower half rich in hydrophobic residues (Hill, 1991). Structural studies on human and rabbit defensins suggest this may be a common structural theme for all defensins (Ganz, et al., 1990). Tachyplesin I (Kawano, et al., 1990) and protegrins (Harwig et al., 1995) which contain two disulphide bonds also adopt an antiparallel P-sheet (so-called hairpin) structure connected by a P-turn.  6  3.2 a-Helical structures Cecropins (Holak et a l , 1988), melittin (Bazzo et al., 1988)  and magainins  (Marion et al., 1988) are typical examples of a-helical structured peptides.  They are  devoid of any cysteine residues and adopt a distinct secondary structure upon interacting with hydrophobic environments like membranes. For cecropin A this consists of two a helical segments joined by a hinge region (Ala-Gly-Pro). The strongly basic N-terminus contains an almost perfect amphipathic helix and the C-terminus contains a more hydrophobic helix (Steiner, 1982; Holak et al., 1988). bend-helix structure.  Melittin adopts a similar helix-  In contrast to cecropin, its N-terminus is hydrophilic and its C -  terminus is hydrophobic. Magainin from amphibian skins also forms an amphipathic a helix (Marion, 1988).  3.3 Extended structures. The group of antimicrobial peptides that are rich in specific amino acids include Bac5 and Bac7 (Frank et al., 1990), PR-39 (Agerberth et al., 1991), prophenin (Harwig et al., 1995) and indolicidin (Selsted et al., 1992).  They all contain a high content of  proline, with Bac5 and Bac7 also rich in arginine, prophenin rich in phenylalanine and indolicidin rich in tryptophan. The presumptive three dimensional structures show that Bac5  and Bac7 contain a series of turn/coil-segments  amphipathic structures (Frank et al., 1990).  interspersed with  extended  Indolicidin has been proposed to form an  extended polyproline type-II helix (Falla et al., 1996).  4. Proposed Mechanism The  ability of polycationic peptides to disrupt the cytoplasmic membrane of  bacteria has been proposed to represent their mechanism o f action (Hancock and Lehrer, 1998). This action is proposed to involve three steps, 1) binding to the cell surface; 2) permeabilization of the outer membrane and then the cytoplasmic membrane; 3) loss of viability of cells involving cell lysis and possible D N A injury (Lichtenstein et al., 1988). The killing is believed to be initiated by the electrostatic interaction of cationic peptides with the negatively charged cell surface.  For Gram-negative bacteria, the  positively charged domain of the cationic peptides binds to the divalent cation binding sites of L P S (Sawyer et al., 1988; Piers et al., 1994). The displacement of the native cations C a  2 +  and M g  2 +  disrupts the structures of the outer membrane, due to the bulky size  of the cationic peptides. This disruption subsequently results in the self-promoted uptake of cationic peptides (Hancock, et al., 1995). For Gram-positive bacteria, the cell wall contains covalently-bound, negatively charged teichuronic acid and carboxyl groups in the amino acids in the peptidoglycan (Hamoond, 1984), and these are probably the initial binding sites for the cationic peptides. The lethal step of killing is believed to involve the interaction of peptides with the cytoplasmic membrane (Lehrer, 1989; Westerhoff et al., 1989; Skerlavaj et al., 1990). The interaction between the peptides and the cytoplasmic membrane is thought to be determined by factors such as the anionic lipid composition of the bacterial membrane, and by the presence  of an electrochemical potential across the membrane.  After  positively charged cationic peptides bind to the negatively charged head groups of lipid, under the influence of a transmembrane potential (oriented internal negative),  the  8  peptides insert into the membrane, and undergo conformation changes to adopt a structured form such as an amphipathic a-helix.  In model systems, peptides, like  cecropin A (Christensen, et al., 1988), magainins (Duclohier, 1994), melittin (Tosteson, 1981) and defensins (Kagan et al., 1990) then aggregate to form multimers, which allow them to form channels or pores with their hydrophobic faces positioned toward the membrane and their hydrophilic faces oriented towards the interior of these channels or pores. In the cytoplasmic membrane of bacteria, this would result in leakage of protons, causing dissipation of the membrane potential, and leakage of other small compounds, thus causing cell death. After the membrane permeability was changed, Lehrer et al (1989) observed the simultaneous  loss  of the  proton  motive  force,  cessation  of biosynthesis of  macromolecules like D N A , R N A and protein, and leakage of intracellular contents, which would presumably be responsible for eventual cell death. Although it seems that channel formation is fundamental to the interaction of peptides with the cytoplasmic membrane, it is still not clear i f channel formation is the mechanism by which these peptides kiir microorganisms. The same factors also seem to regulate the selectivity of cationic peptides for bacterial membranes over eukaryotic cell membranes. 1) The compositions of their membranes are quite different with bacterial membranes predominantly containing negatively charged lipids such as phosphatidylglycerol and cardiolipin, while the eukaryotic cell membrane  is largely composed of zwitterionic lipids such as  phosphatidylcholine and sphingomyelin. 2) Eukaryotic cell membranes are rich in  9  cholesterol, which may inhibit membrane insertion. 3) Bacterial cells have large, interiornegative transmembrane potentials of around -140  m V whereas eukaryotic plasma  membranes have membrane potentials of only - 2 0 m V or so. There are a few cationic peptides which have been proposed to function through non-membranous mechanisms, including apidaecin from honeybees (Casteels, et al., 1989), thanatin and attacin. Apidaecin was shown to lack the ability to permeabilize the membrane and the all-D-enantiomer of apidaecin was completely devoid of antibacterial activity (Casteels and Tempst, 1994). Apidaecin has no effect on Gram-positive bacteria and it does not show homology with any other cationic peptides.  It was suggested that  the action of apidaecin on bacteria involved a stereoselective recognition of a chiral cellular target.  Correspondingly, it has been reported that attacin inhibits synthesis of  outer membrane proteins in E. coli through specific  interference  with omp  gene  transcription (Carlsson, et al., 1991; Hultmark et al., 1983).  5. Structure and Function Relationships A variety of studies have examined the relationship between chemical structure and antimicrobial activity. There are several structural requirements that have been found to be of importance in determining antimicrobial activity. In general, the results clearly indicate a requirement for amphipathic structures and positive charges.  Although it is  speculated that more positive charges will facilitate the interaction of cationic peptides with the target organism, there is no absolute relationship between the number of positive charges and activity.  The position of specific positive charges may be of importance  10  (Blondelle and Houghten, 1991). Increasing the a-helical content appears to increase the antimicrobial activity of a-helical peptides (Andreu et al., 1985; Steiner et al., 1988; Frohlich and Wells, 1991; Blondelle and Houghten, 1991). For the a-helical peptides, enantiomers have equal activity (Besalle et al., 1990; Wade et al., 1990), showing that chirality is not important. For the disulfide-bonded peptides, intra-chain disulphide bonds are essential for antimicrobial and cytotoxic activity (Lehrer, 1993).  The disulfide structure of the  defensins is essential for structural stability and potency (Ganz, et al., 1987; Selsted et al., 1983). Tachyplesin I is a 17-residue peptide from horseshoe crab hemocytes. It contains two disulfide bonds, and studies showed that the deletion of these disulfide bridges caused a major decrease in all activities (Tamamura et al., 1993; Matsuzaki, et al., 1993). Protegrins are p-sheet structured peptides, containing 16 to 18 residues and two intramolecular disulfide bonds. Both intra-chain disulfide bonds are required for the antiparallel P-sheet conformation in membrane-mimetic environments and for optimal antimicrobial activity, especially in media containing NaCl, with at least one disulphide bond being needed for any activity (Sylvia et al, 1996; Yasin, et al., 1997; Qu, et al., 1997). Deletion of two disulfide bridges in protegrin-1 caused a significant decrease in antibacterial activity against both Gram-negative and Gram-positive bacteria, as well as antiviral activity (Tamamura, et al., 1995). The above results all indicate the importance of the structure maintained by the disulphide bonds for the antimicrobial activity of these peptides. A cationic cluster flanked by a hydrophobic cluster appears to be a common  11  structural feature shared by tachyplesin, protegrin and human defensin (Kini and Evans, 1989; Aumelas, et al., 1996). The above observations and future studies involving more specific changes may provide guidelines for the design of potent synthetic antimicrobial peptides.  B. Loop-Structured Cationic Peptides and Bactenecin. Recently, a few cationic peptides with only a single disulphide bond forming a loop structure, have been identified (Morikawa, et al., 1992; Simmaco et al., 1993; Clark et al., 1994; Suzuki, et al., 1995; Fehlbaum, et al., 1996). According to the source from which they were derived, they can be sorted into two groups. Their sequences are shown in Table I.  The first group were isolated from frog (Rand) skin, namely the brevinin  family (brevinins 1, I E , 2, 2E) (Morikawa, et al., 1992; Simmaco, et al., 1993), esculentin (Simmaco, et al., 1993), the rugosin family (rugosins A , B , C) (Suzuki, 1995), and  Table I: Loop-Structured Cationic Peptides Name  Source  Amino A c i d Sequence  Brevinin-1  Skin, Rana brevipodaporsa  FLPVLAGIAAKVVPALFC KITKKC-OH  Brevinin-2  Skin, Rana brevipodaporsa  GLLDSLKGFAATAGKVL QSLLSTASCKLAKTC-OH  Brevinin-IE  Skin, Rana esculenta  FLPLLAGLAANFLPKIFC KITRKC  Con't  12  Table I Loop-Structured Cationic Peptides (Con't) Brevinin-2E Skin, Rana esculenta  GIMDTLKNLAKTAGKG ALQSLLNKASCKLSGQC  Esculentin  Skin, Rana esculenta  GIFSKLGRKKIKNLLISG LKNVGKEVGMDVVRTG IDIAGCKIKGEC  Ranalexin  Skin, Rana catesbeiana  FLGGLIKIVPAMICAVTK KC-OH  Rugosin A  Skin, Rana rugosa  GLLNTFKDWAISIAKGA GKGVLTTLSCKLDKSC  Rugosin B  Skin, Rana rugosa  SLFSLIKAGAKFLGKNLL KQGAQYAACKVSKEC  Rugosin C  Skin, Rana rugosa  GILDSFKQFAKGVGKDL IKGAAQGVLSTMSCKLA KTC  Thanatin  B-dodecapeptide  Insect, Podisus  GSKKPVPIIYCNRRTGKC  maculiventris  QRM  Bovine neutrophil  RLCRIVVIRVCR  Sheep myeloid cells  RICRIIFLRVCR  (Bactenecin) s-dodecapeptide  Cysteine residues involved in disulphide bond formation are underlined.  13  ranalexin (Clark, 1994). Thanatin (Fehlbaum, 1996) was isolated from insects, but shows similarity to with the frog skin peptides. amino acids to 46 amino acids.  The lengths of these peptides ranges from 20  Brevinins, rugosins, ranalexin and thanatin share  significant amino acid sequence similarity. The sequence of esculentin is not related to any of the known cationic peptides.  This group of peptides all contain a single C -  terminal intramolecular disulfide bond which forms a cationic loop. Except for thanatin, the loop contains five residues at the C-terminal end. Thanatin has a loop of six residues and 3 extra amino acids at the C-terminus outside the ring. These frog skin peptides exhibit different potencies against bacteria.  Most of  these peptides have antimicrobial activity against both Gram-negative (E. coli) and Grampositive (S. aureus) bacteria. Rugosin A , rugosin B and brevinin 1 are selectively active against Gram-positive bacteria.  Brevinin I E was shown to have high lytic activity.  Thanatin was also shown to be fungicidal, and did not exhibit hemolytic activity. Few studies of the antimicrobial mode of action have been done on these peptides, with the exception o f thanatin.  Brevinin-2 and esculentin have an N-terminal region  which can form an amphiphilic a-helix, and was suggested to be involved in membrane permeabilization (Kreil, 1994).  Ranalexin has a structure similar to polymyxin B and  was proposed to function like polymyxin B (Clark, 1994).  It was suggested that the  cytoplasmic membrane is not the target of thanatin, and thanatin is not a pore-forming peptide (Fehlbaum, 1996).  A n all-D-enantiomer of thanatin is nearly inactive against  Gram-negative bacteria, but maintains activity against some Gram-positive bacteria and  14  all fungi. This suggested that more than one mechanism might be involved in the antimicrobial activity of thanatin. The second group of loop-structured peptides contains bactenecin (also called bovine  dodecapeptide)  from  bovine  neutrophils  (Romeo  et  al., 1988)  and  ovine  dodecapeptide from sheep myeloid cells (Bagella, et al., 1995). Both are 12 amino acids long, including four arginine residues, two cysteine residues, and 6 hydrophobic residues. Ovine dodecapeptide has four conserved hydrophobic residue-substitutions compared to bovine dodecapeptide, including one phenylalanine in the ring.  These peptides are the  smallest known cationic antimicrobial peptides. The cysteine residues, close to either end form a disulphide bond to make a loop. The mode of synthesis of these peptides involves prepropeptides, a precursor which identify these peptides as belonging to the cathelicidin family. Cathelicidins are a novel protein family, which have highly conserved N-terminal preproregions, and variable C-terminal antimicrobial cationic peptide regions (Zanetti, et al., 1995).  They  have been identified in mammalian myeloid cells, including bovine (Storici et al., 1996), porcine (Tossi et al., 1995), rabbit (Levy, et al., 1993), sheep (Bagella, et al., 1995) and human (Cowland et al., 1995) myeloid cells. Bactenecin was previously found to be active against E. coli and S. aureus, and strongly cytotoxic for rat embryonic neurons, fetal rat astrocytes and human glioblastoma (Radermacher, et al., 1993). However, little is known about its antimicrobial mechanism and whether it shares a common killing mechanism with other antimicrobial peptides, or  15  if it has a distinct mode of action due to its unique compact structure (c.f. the silk moth peptide cecropin which is a 26-amino acid amphipathic a-helix).  C. Therapeutic Potential The earliest peptide antibiotics that were used extensively in human medicine (mainly topical therapy) were the gramicidins and polymyxins.  The lantibiotic  bacteriocin nisin is currently used as food preservative (Delves-Broughton et al., 1996). MSI-78, a 22 residue magainin analogue has recently completed human Phase III clinical trials, showing equivalent efficacy to oral ofloxacin vs. polymicrobic infections of individuals with diabetic foot ulcers (Hancock, 1997). Another promising prospect for cationic peptides would be plant and fish biotechnology in which cationic peptides are engineered by transgenic techniques into host organisms to permit enhanced bacterial disease resistance in crops and fish (Hancock and Lehrer, 1998). There are many interesting features of cationic peptides as potentially novel antibiotics. Besides broad antimicrobial activity, there are two other main antibacterial activities of these peptides, namely their ability to bind to LPS and act as an antiendotoxin (Gough et al., 1996), and their ability to act synergistically with conventional antibiotics as enhancers. Resistance against antimicrobial peptides may not develop easily. Some bacterial strains that show resistance to cationic peptides have been identified, e.g. Burkholderia cepacia, Proteus sp. and Brucella sp. They have developed resistance intrinsically rather than by exposure to the peptides.  Since these cationic peptides are gene-coded, and  16  synthesized as precursors that undergo posttranslational modifications to become active, their production by gene engineering becomes possible. While researchers continue to isolate and characterize new cationic peptides from nature, the study o f analogs, as well as studies o f structure:function relationships have become the focus in new drug discovery efforts.  D . A i m s of T h i s Study Bactenecin was selected as the model peptide in this study. structured cationic peptides have not been well studied.  The group of loop-  The unique structure of  bactenecin among known peptides (e.g. small size, single disulphide bond and loop structure) makes it an interesting peptide to study. The general aim o f this study was to investigate the relationship between the structure and activity o f bactenecin. The mode of action and antimicrobial mechanism o f bactenecin was examined and several bactenecin derivatives were designed,  aimed at both discovering a superactive candidate and  understanding the mode o f action.  17  MATERIALS AND METHODS  A . Bacterial Strains and Growth Conditions A l l bacterial strains used in these studies are listed in Table II. Bacterial strains for cloning purposes included E. coli D H 5 a , E. coli B L 2 1 , S. aureus K147. A l l strains were grown in Luria broth ( L B N S : 10 g/L tryptone and 5 g/L yeast extract, 5 g/L NaCl), or L B N S supplemented with 10 pg/ml chloramphenicol or lOOpg/ml ampicillin. Bacterial strains for antimicrobial activity testing were grown in no salt Luria broth (10 g/L bacto-tryptone and 5 g/L bacto-yeast extract).  Streptococcus strains were  grown in Todd Hewitt broth (500 g/L beef heart infusion, 20 g/L bacto neopeptone, 2 g/L bacto dextrose, 2 g/L sodium chloride, 0.4 g/L disodium phosphate, 2.5 g/L sodium carbonate).  Both L B and Todd Hewitt broth were purchased from Difco Laboratories,  Detroit, Michigan.  B. Chemicals Polymyxin B and 1-N-phenylnaphtylamine (NPN) were purchased from Sigma (St. Louis, M o ) . 3,3-Dipropylthiacarbocyanine (DiS-C3-(5)) was from Molecular Probes (Eugene, Or). Dansyl polymyxin B was synthesized as described previously (Schindler andTeuber, 1975). The lipids l-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine and  l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol  (POPC)  (POPG) were purchased from  Northern Lipids Inc. (Vancouver, B C , Canada).  18  NO 00 ON  NO ON  00 ON  > ^  CN CN OO 00 ON Os  1 - 1  ON ON  oo oo ON  IH  ON  rt  rt  c §  00 ON  CO„  GO fl  o  Ss  KS  rt  <D  ID  of  GO*  to (L)  cu  oo  5s.  • *-S fl" fl fl  o  s i fl-  CX  u  OH  l-i  D  00  '-+-> a  <+H  oo  fl. X <D  fl  "rt o  £ M° * o  fl  2  (D  T3  o  11  !•  ^  ° "S  s  •S c s  co 00 OO  ^  S3 ON  to ^  a,  o NO  i4 N  CU  S ^  <2 3o to 3 cu  s  oo  oo fl 13 ~  rfl 4) 3  o oo --j CN fl -fl. ^ _H t' 5CO l-l  H  ^  "rt  2  [fl  !<  a a to s CJ o o  Ci_ 00  .3 ^  -R  Is•a .2  fl* O  g%  co  o  O  '3  2  1  «I s  fl  fl  in S ON  00  S  - to  "S — 1  00  1  ^  fl  CO  <D  oo fl  GO  fl fl GO Q fl i-i Gl-lO o fl <8 <D Co 5) fl fl^ ^ S3 P-t 00 o .2 to co  00  00  <u o  o  'oo  00  CO  s  CN  l-l  o fl  >  GO  •a  o  u  • I—I  13 13  o  c  <o  CN  °  ^  CJ  O  —<  w OO  H  « m NO  co U  U  5T  to  o  -^CN •s? CN  §r z  "53  CN ON  s u o o  CN  o  m  S  -IB r-H  ^ &0 <  CJ  &, CN  •S ^  co  U  OS  CD  C  i-i  o  O  =3  O GO  fi  C '-*-> o  o  »  o  CD  CD  "o  CD  ~CD  o fi <u  o  O U H  <  C<H  CD  O O  O O H  U  tH  U  H  CD  CD  "o  CD  CO  co  o  13 o  ;a  CD  o  CD  CO  fi B o U  O  •c  ' f i • r-H  o  co  u  CD  u  U  c  ~CD  CD  o  i—i 13 o  2  • i—i  »5  CD  u <u  g  #  '>3 -  CU  e  CC  1-  cu  cu « P9  ^  CN  S CN U  CO  fi 'c3  is cc  O  ° ^  ^  o s o £ CU  s H  h3  <  sr <u R  R  O  3  O  £ s  <U  cu R cu CJ  <3  -Ci CU R  b  a  =o 3  cj  CJ  ON  to  .3 0=5  vo  O ON  cj  o  C3  CJ  »3  CO  <  IS  S3 <  s  CJ CJ  O  cj O  Si  3  o  C. Recombinant Expression of Peptides C.I. D N A Manipulations General D N A techniques such as plasmid preparations from E. coli, agarose gel electrophoresis, D N A ligation, E. coli D H 5 a transformation and restriction enzyme digestion were performed as described in Ausubel et al.. (1987) and Sambrook et al. (1989). and  Slot lysis gel electrophoresis was according to Sekar (1987).  D N A restriction  modifying enzymes and D N A ligase (Bethesda Research Laboratories (BRL),  Burlington, M A ) were used according to the manufacturers' methods.  D N A fragment  isolations from agarose gels were performed using the Geneclean kit (Bio 101 Inc., L a Jolla, C A ) according to the manufacturer's protocol.  D N A sequencing was done using  Taq Dye Deoxy Terminator Cycle Sequencing Kit on an A B I 370A automated D N A sequencer ( A B I , Foster City, C A ) . Plasmid D N A for sequencing was purified using Qiagen columns  (Qiagen Inc., Chatsworth, C A ) , by following  the  manufacturer's  protocol.  C.2. Electroporation The method for electroporation of S. aureus RN4220 was based on that of Kraemer and Landolo (1990). OD =0.3-0.8. 600  Briefly, cells were grown in 100 m l L B N S broth to an  Harvested cells were washed twice with 500 m M sucrose and placed on  ice for 15-30 min.  The cells were finally resuspended in 10 m l of 500 m M sucrose.  Then, 140 ng plasmid D N A were mixed with 160 p i cells in a cuvette, and chilled on ice for 1 min.  Electroporation was carried out using a BioRad (Mississauga, Ont) Gene  Pulser set at 200 ohm, 1.8 k V . Immediately after charging, 1 ml S M M P medium (2.8%  21  (w/v) Bacto Pennassay Broth with 275 m M sucrose, 11 m M maleic acid, 11 m M M g C l  2  and 2.5% (w/v) B S A ) was added and the mixture incubated on ice for 15 min before plating on L B N S plates with 10 pg/ml o f chloramphenicol.  C.3. Oligonucleotide Synthesis and Purification The amino acid sequences o f indolicidin, apidaecin and bactenecin were used to design three oligonucleotide sequences according to the  S. aureus codon preference  (Wada et al., 1992) (Fig 1 & F i g 2B). Both coding strands and template strands for these cationic peptides were made. The two complimentary strands were annealed by mixing 1 mg/ml D N A o f each strand in the annealing buffer (20 m M T r i s - H C l p H 7.5, 10 m M MgC12 and 50 m M NaCl). The mixture was heated at 90 °C for 2 min, then cooled down gradually. The annealed double stranded D N A was quantitated by U V absorbance at 280 nm and used directly for ligation. Primers 1 and 2 (Fig. 2C) for D N A sequencing after cloning into pRIT5 were also made according to the upstream and downstream sequences of the multiple cloning site o f pRIT5. In order to be able to clone the peptide genes into vector pSP72, a third primer (Fig. 2C) primer 3 was made to incorporate an EcoRV site. The bactenecin peptide gene fragment was obtained by P C R (94 °C l m i n , 62 °C 2 min, 72 °C 1-1.5 min 20 cycle, 72 °C 10 min) from vector pRIT5 using primers 2 and 3, and subcloned into pSP72. A l l oligonucleotides were synthesized on an Applied Biosystems Inc., Model 380B D N A synthesizer and purified according to manufacturer's instruction.  22  Apidaecin Ia  GNNRPVYIPQPRPPHPRI  Indolicidin  ILPWKWPWWPWRR  Bactenecin  Figure 1: A m i n o  RLCRIVVIRVCR  acid sequences of the three recombinantly expressed cationic peptides.  The charged amino acids are in bold, the characteristic amino acids for each specific peptide are underlined.  23  A. Construction of Cationic Peptide Genes for Cloning into pRIT5 AATTC C ATG G G TAC EcoRI Start  Cationic Peptide GeneSequence  TAA C TAA G TAA GAGCTC G A T T G ATT C ATT C T C G A G C A G C T Stop Stop Stop Sst I Sail  B. Three Cationic Peptide Gene Sequences (Single Strand 5'-3') Apidaecin GGT A A T A A T A G A C C A GTT T A T A T T C C A C A A C C A A G A C C A C C A C A T CCA A G A TTA Indolicidin A T A TTA C C A T G G A A A T G G C C A T G G T G G C C A T G G A G A A G A Bactenecin A G A T T A TGT A G A ATT GTT GTT ATT A G A GTT TGT A G A  C. Primers for Sequencing and PCR Primer 1: A C G T A A C G G CTT C A T C C A A A G Primer 2: A G C T A T G A C C A T G A T T A C G C C Primer 3: A A A G A C G A T A T C G G G G A A TTC C  Figure 2: Sequences of oligonucleotides. Panel A shows the construction of the oligonucleotides synthesized for cloning into pRIT5. Panel B shows the corresponding cationic peptide gene sequences. Primers 1 and 2 in panel C were designed for sequencing. Primers 2 and 3 were used to engineer an EcoR I site into an EcoR V site so that the cationic peptide genes could also be cloned into pSP72.  24  D. Purification of the Recombinant Peptides D.l. Electrophoresis  SDS-PAGE was performed as previously described (Hancock and Carey, 1979). Acid-Urea gels (AU-PAGE) were prepared according to the method of Panyim and Chalkey(1969). D.2. Purification of the Protein A/Cationic Peptide Fusion Protein  Two litre LBNS cultures of S. aureus strains harboring the various plasmids, pPA-bactenecin, pPA-apidaecin and pPA-indolicidin, were grown at 37 °C to an OD =1.5-1.8. Cells were removed by centrifugation, and the supernatant was collected, 600  adjusted to pH 7.6 and passed over an IgG-Sepharose-6 Fast-Flow column (Pharmacia). The column was loaded, washed and fusion protein was eluted according to the manufacturer's protocol. To ensure fusion proteins were eluted correctly, samples were taken to run on SDS-PAGE.  Fractions containing pure fusion proteins were pooled,  lyophilized and the residue resuspended in 70 % formic acid for the CNBr digestion. D.3. Cleavage of Cationic Peptides f r o m Protein A  Cationic peptides were cleaved from protein A by CNBr which cleaved at internal methionines. The cleavage reaction was carried out in 70 % formic acid and 1M CNBr in the dark at room temperature overnight. The reaction was quenched by diluting to 5 % formic acid with distilled water. The sample was then lyophilized. The dried sample was resuspended in 1 % acetic acid for loading onto a PI00 column. A U - P A G E was run to ensure that cationic peptides were cleaved properly from the carrier.  25  D.4. Purification of Cationic Peptide by P 1 0 0 Column Chromatography The sample after cleavage was applied to a Bio-gel polyacrylamide PI00 column (Biorad). The PI00 column was packed according to the manufacturer's protocol. Two bed-volumes of 1 % acetic acid were passed over the column by gravity feed. One-ml fractions were collected. Fractions were monitored at 280 nm, and samples taken every 5 fractions for analysis by A U - P A G E .  Fractions containing peptides were pooled,  lyophilized and resuspended in distilled water. D. 5. Expression of Fusion Gene in E. coli One single colony of E. coli harboring plasmid pSP72/bactenecin was picked and grown overnight at 37 °C in L B broth containing 100 pg/ml ampicillin. Fresh L B broth (5 ml) with 100 pg/ml ampicillin was inoculated with the overnight culture and grown for an additional 3-4 hours until O D  600  = 0.6-1. The culture was then induced with 1 m M  IPTG. The cells were harvested 2-3 hours later after induction, and resuspended in 100 pi T E buffer (10 m M Tris-HCl, 1 m M EDTA). A 10 pi sample was removed and mixed with 2-fold concentrated SDS-PAGE sample buffer (4 % SDS, 0.2 M Tris pH 6.8, 20 % glycerol, 8 % p-mecaptoethanol). The mixture was heated at 95 °C for 5 min. A sample of 5 pi was loaded on a 12 % SDS-PAGE to check the production of fusion protein.  E. Peptide Preparation E . l . Synthesis of Peptides. Peptides were synthesized by Fmoc (N-(9-fluorenyl) methoxycarbonyl) chemistry by the Nucleic Acid/Protein Service unit at the University of British Columbia using an  26  Applied Biosystems, Inc. (Foster City, C A ) Model 431 peptide synthesizer.  The  amidated peptides were provided by Micrologix Biotech Inc., Vancouver, B.C., Canada. Gramicidin S was purchased from Sigma (St. Louis, MO), and Gram474 and Gram4112 were provided by the Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton Alberta, Canada. A l l the peptide sequences and their sources are listed in Table III and Table IV. E.2. Refolding of bactenecin and its derivatives. The purchased bactenecin and its derivatives were in their fully reduced form. After a series of trials to determine the optimal strategy, the disulphide bond was formed by air-oxidation in 0.01 M Tris buffer, pH 7.2, at 23 °C for 24 h. The concentration of peptides was kept below 100 pg/ml in the oxidation buffer to minimize the formation of multimers. After oxidation, the solutions were frozen and lyophilized. The dried sample was resuspended in 0.3 %  (v/v) T F A and centrifuged to remove any debris before  loading on a column. E . 3 . Peptide Purification A reversed phase Pep RPC HR5/5 (Pharmacia; Quebec, Canada) column was used to purify the disulphide-bonded bactenecin and derivatives from the multimer byproducts. The column was equilibrated with 0.3 % (v/v) aqueous trifluoroacetic acid and eluted with a slow gradient of acetonitrile (0.1 % acetonitrile per minute) in 0.3 % T F A at a flow rate of 0.7 ml/min. The absorbance was monitored at 220 nm. containing the desired product were pooled and lyophilized.  Fractions  Matrix-assisted laser  desorption/ionization (MALDI)-mass spectrometry and A U - P A G E (Spiker, 1980)  27  Table III: Amino Acid Sequences of Bactenecin and its Derivatives  Peptide  Amino Acid Sequence  Number of Amino Acids  Number of Positive Charges at pH 7  RLCRIVVIRVCR  12  4  Bac2A-CN  RLARIVVIRVAR-CONH,  BacS-CN  RLSRIVVIRVCR- CONH"  Bac2S-CN  RLSRIVVIRVSR-  12 12 12 12  5 5 5 4  12 12 12 12 12 14 14 14  5 4 5 5 4 6 6 6  13 13  4 4  Native Bactenecin  Linear  Bac2S  CONH,"  RLSRIVVIRVSR  Positive Charge BacR, P - C N BacR, P  RRCPIVVIRVCR-  RRCPIVVIRVCR  Bac2I-CN BacP, 2 R - C N  CONH,  RICRIVVIRCIR-  CONH,  RLCPRVRIRVCR- CONH,  Bac3K, P Bac3R, P Bac3R, P , ( V ) Bac2R  KKCPIVVIRVCK RRRCPIVVIRVCRR RRRLCPIVIRVCRR RRLCRIVVIRVCRR  Hydrophobicity BacP BacW  RLCRIVPVIRVCR RLCRIVWVIRVCR  Others BacW, 2R  15 Underlined residues are amino acids different from native bactenecin. PxRLCRIVWVIRVCRR  6  28  Table IV: Amino Acid Sequences of other Cationic Peptides Used in This Study  Peptides  Amino Acid Sequence  Number of Amino Acid 26 26  Number of Positive Charges 7 5  KWKLFKKIGIGAVLKVLTTGLPALKLTK  28  7  CP 26 CP 27 (CEME) CP 28 (CEMA) CP 29  KWKSFIKKLTSAAKKVVTTAKPLISS KWKLFKKIGIGAVLKVLTTGLPALIS  KWKSFIKKLTTAVKKVLTTGLPALIS  26  6  CP 10CN CP 11CN  ILPWLWPWWPWRR-CONH2 ILKKWPWWPWRRK-CONH2  13 13  3 5  10 14 14  2 4 4  Gramicidins F P V O L F P V O L Gram474 Y P V K L K V Y P V K L K V Gram4112 Y P V K L K V Y P L K V K L O: Ornithine Residues underlined are D-amino acids.  were used to confirm that the disulphide bond was properly formed, and a pure product obtained. E.4. Peptide Concentration Determination Concentrations of gramicidin S and analogs were determined by the dried weight of powder. E.4.a. Amino Acid Analysis Concentrations of bactenecin and its derivatives were determined by amino acid analysis at the N A P S unit at U B C .  E. 4.b. Dinitrophenylation. Concentrations of CP26, CP27, CP28, CP29,  CP10CN  and C P 1 1 C N were  estimated by the dinitrophenylation assay, which measures the presence of free amino groups (Bader and Teuber, 1973).  F . Structural Studies F . l . Circular Dichroism A  Jasco (Japan) J-720 spectropolarimeter was used to measure the circular  dichroism ( C D ) spectra of bactenecins (Falla et al., 1996). The data were collected and analyzed by Jasco software.  Liposomes P O P C / P O P G (7:3) were prepared by the freeze-  thaw method to produce multilamellar vesicles as described previously (Mayer et al., 1985), followed by extrusion through 0.1 m m double stacked Nuclepore filters using an extruder device (Lipex Biomembranes, Vancouver, B C Canada), resulting in unilamellar liposomes.  C D measurements of peptide solutions were taken at a final concentration of  50 p M peptide in 10 m M sodium phosphate buffer, pH7.2. T F E at a final concentration 60 % (v/v), S D S (final concentration 10 m M ) or liposomes (final concentration 100 u M lipid) were added and incubated at room temperature for 10 min before the C D measurement.  F.2. Computer Modeling Computer  modeling  of peptides  was  done  using  the  Insight  II program  (Biosystems, San Diego, C A ) . The secondary structure was modeled based on energy minimization for 4000 iterations.  Three steps of minimization were performed to reach  the minimal total energy state: 1) Steepest descent; 2) conjugate gradient; 3) V A 0 9 A .  30  G. Antimicrobial Activity and Hemolytic Activity G . l . Minimal Inhibitory Concentration Determination The minimal inhibitory concentrations (MICs) o f peptides were determined by a two-fold microtitre broth dilution method modified from that o f Steinberg et al (1997). Using the classical method (Amsterdam, 1991), higher concentrations of peptides tend to precipitate in the L B broth, thus the concentrations of peptides in the sequential wells are not accurate. Also the peptides stick to the most readily available (tissue-culture treated polystyrene)  96-well  microtitre plates.  Therefore the 2-fold dilution series were  performed in Eppendorf tubes (polypropylene).  Serial two fold dilutions of  peptides  ranging from 640 pg/ml to 1.25 pg/ml were made in 0.2% B S A , 0.01% acetic acid buffer. Ten pi o f each concentration were added to each corresponding well o f a 96-well microtitre plate (polypropylene cluster; Costar Corporation, Cambridge, M A ) . Bacteria were grown overnight and diluted 10" into fresh L B broth or Todd Hewitt broth for 5  Streptococcus.  One hundred p i of broth containing about 10 -10 4  5  C F U / m l o f tested  bacteria were added to each well. Plates were incubated at 3 7 ° C overnight. The M I C was taken as the concentration at which greater than 90% o f growth inhibition was observed.  G.2. Human Red Blood Cells Lysis Assay Human  red blood cells  ( R B C ) were  centrifuged to remove the buffy coat. centrifuged at 1500 xg for 5 min each.  freshly  collected  with heparin, and  R B C were then washed in saline 3 times and The erythrocytes were finally resuspended in  saline to packed cell volume at the ratio o f 25/1.  Serial 2-fold dilutions of test peptides  31  were made in a microtitre plate with 100 pi saline per well. Fifty microlitre o f R B C were added.  Plates were covered and incubated with rocking at 37 °C. Readings o f lysis or  agglutination o f R B C were taken after 4 and 24 h. Concentrations at which 90% lysis or agglutination  was  observed  were  taken  as  the  hemolytic  or  hemagglutination  concentrations.  H . Membrane Permeabilization Assays H . l . Isolation of L P S E.coli UB1005 L P S was prepared according to the phenol-chloroform-petroleum ether extraction method (Gerhardt, 1994).  Briefly, two six-liter cultures o f E. coli  UB1005 were grown to late logarithmic phase. Cells were harvested and washed sequentially with 150 ml cold deionized water once, 90 ml cold methanol once and 90 ml cold acetone twice. DNase (100 pg/ml) or RNase (25 pg/ml) were added. The pellet was then air-dried overnight. Dried cells were extracted with 50 m l P C P (mixture of 40 ml phenol, 100 m l chloroform, and 160 ml petroleum ether) twice by homogenizing at maximum speed for 30 s. Supernatants were collected and filtered through Whatman no. 1 paper, and concentrated under a stream of N  2  gas at 50 °C until the L P S precipitated.  After centrifugation, the pellet was washed three times with 20 m l methanol, and then dried for 2 hr. The dried L P S was resuspended in 24 m l water and ultracentrifuged at 200,000 x g for 4 hr. The final product was resuspended in water again and lyophilized. A homogenizer was used to resuspend the pellets after each step.  H.2. Dansyl-polymyxin B displacement assay The dansyl-polymyxin B displacement assay (Moore et al., 1986) was used to determine the relative binding affinity of peptides for L P S .  H.3. Outer Membrane Permeability Assay The ability of peptides to permeabilize the outer membrane was determined by the N P N assay of L o h et al (1984).  H.4. Inner Membrane Permeability Assay Cytoplasmic membrane permeabilization was determined by using the membrane potential sensitive carbocyanine dye DiS-C3-(5) (Sim et al, 1974).  The mutant E. coli  D C 2 with increased outer membrane permeability was used so that DiS-C3-(5) could readily reach the cytoplasmic membrane.  Fresh L B medium was inoculated with an  overnight culture, and incubated at 37 °C with shaking, and mid-logarithmic phase cells (OD  6 0 0 n m  = 0.5 - 0.6) were collected. The cells were washed with buffer (5 m M H E P E S , 5  m M glucose, p H 7.2) once, then resuspended in the same buffer to an O D  6 0 0  of 0.05. The  cell suspension was incubated with 0.4 p M DiS-C3-(5) until DiS-C3-(5) uptake was maximal (as  indicated by a stable reduction in fluorescence  due to  fluorescence  quenching as the dye became concentrated in the cell by the membrane potential), and 100 m M K C I was added to equilibrate the cytoplasmic and external K  +  concentration.  One-ml of cell culture was placed in a 1-cm cuvette and the desired concentration of tested peptide was added. The fluorescence reading was monitored by using a PerkinElmer model 650-10S fluorescence  spectrophotometer (Perkin-Elmer Corp. Norwalk,  Conn), with an excitation wavelength of 622 nm and an emission wavelength of 670 nm.  33  The maximal increases in fluorescence  due to the disruption of the cytoplasmic  membrane by different concentrations of cationic peptides were recorded. A blank with only cells and the dye was used to subtract the background. Control experiments (chapter 3) titrating with valinomycin and K showed that the increase in fluorescence was directly +  proportional to the size of the membrane potential and that a buffer concentration of 100 mM KC1 prevented any effects of the high internal K concentration and corresponding +  opposing chemical gradient.  34  RESULTS  CHAPTER I: RECOMBINANT EXPRESSION OF CATIONIC PEPTIDES  A. Introduction Polycationic peptides were originally obtained either by isolation from the host organism or by chemical synthesis. Either method is costly and difficult to scale up. To allow for large scale purification, two bacterial expression systems were developed successfully in Hancock's laboratory. The first system utilized a protein A fusion protein secreted by S. aureus (Piers, 1993). The second one involved a small replication protein R e p A from E. coli, a synthetic cellulose binding domain and a hexa histidine domain as fusion partner, and expressed in E. coli (Zhang et al., 1998).  Recombinant peptide  production will allow future screening of interesting variants created by semi-random mutagenesis, which will facilitate the structure and function relationship studies. It will also allow incorporation of heavy atoms to do 2 D - N M R studies. Three cationic peptides were selected for study due to their unique amino acid sequences (e.g. rich in Trp or Pro) which suggested that they probably have unique structures, namely apidaecin (Casteels, 1989) from honeybees, bactenecin (Remeo  1988)  and indolicidin (Selsted, 1992) from bovine neutrophils (Fig 2). Each has its own distinct characteristics.  Apidaecin is 18 amino acids long and rich in proline. Bactenecin is 12  amino acids long and contains two cysteines which form one disulfide bond and maintain the peptide in a cyclic structure.  Indolicidin is 13 amino acids long and rich in  tryptophan.  35  The objectives of the work described in this chapter were to produce these three active cationic peptides in bacterial expression systems and obtain them in relatively pure and biologically active forms.  B. Recombinant Expression of Three Small Cationic Peptides in S. aureus Plasmid pRIT5 is a shuttle plasmid between E. coli and S. aureus, allowing cloning procedures to be carried out in E. coli and gene expression in S. aureus. Since the coding sequences for mature apidaecin, indolicidin and bactenecin are small (36-54 nt), they can be made as oligonucleiotides.  Three oligonucleotides were designed and  synthesized according to the amino acid sequences of peptides; an EcoRI site and a start codon were included at the 5'-end and a Sail site and stop codons were included at the 3'end (Fig. 2 A and 2B).  These oligonucleotides were ligated into the polylinker cloning  sites between EcoRI and Sail on plasmid pRIT5 and transformed into E. coli D H 5 a . After clones with inserts were obtained, sequencing was done to confirm that they contained the correct sequences of peptide genes. Then  plasmids  pPA-apidaecin, pPA-indolicidin  and  pPA-bactenecin  were  electroporated into S. aureus. Plasmid pRIT5 contained a protein A signal sequence just upstream of the protein A gene, allowing the fusion protein to be exported to the external medium which would reduce proteolytic degradation of the fusion protein.  Strains  containing the plasmid of interest were grown overnight,  growth  samples  of the  supernatants were analyzed by S D S - P A G E (Fig. 3). Protein A has a molecular weight o f 32 k D (Fig. 3, lane 1).  The fusion protein with the cationic peptide will have higher  36  Figure 3:  P r o d u c t i o n o f protein A / c a t i o n i c peptide fusion proteins i n S. aureus.  S. aureus strains w h i c h harbored pRIT5 w i t h specific inserts were g r o w n overnight i n L B broth. Fifteen m i c r o l i t r e o f culture supernatant from each strain were taken a n d r u n o n S D S - P A G E . L a n e s : 1, p r o t e i n m o l e c u l a r w e i g h t markers as indicated; 2, p r o t e i n A secreted b y S. aureus; 3, protein A / a p i d a e c i n fusion protein; 4, protein A / b a c t e n e c i n fusion protein; 5, protein A / i n d o l i c i d i n fusion protein.  37  molecular weight than protein A. Bands with higher molecular weight fusion proteins of protein A-apidaecin (lane 2), protein A-indolicidin (lane 4) and protein A-bactenecin (lane 3) were detected which showed these three fusion proteins were stably produced and secreted out of S. aureus. After the production of the desired products was confirmed, large scale purifications of fusion proteins were carried out by IgG affinity column chromatography. In order to produce active cationic peptides, they were cleaved from protein A with CNBr which cleaved at methionine residue. After the cleavage, the mixtures were run on acidurea gels and the cationic peptides apidaecin, indolicidin and bactenecin were detected (Fig 4). Two bands of bactenecin were detected: the upper band was believed, and confirmed by mass spectrometry to be a multimer, and lower band to be intra-chain disulphide-bonded bactenecin. After the cleavage, purification of cationic peptides from CNBr-digested protein A fragments was done by running the CNBr-digested mixture through a size exclusion Bio-gel PI00 column (Fig 5A, 6A, 7A). The elution profiles showed that all three cationic peptides were eluted in the last (third peak), and further A U gel analysis (Fig. 5B, 6B, 7B) showed that the three cationic peptides were reasonably pure after the size exclusion purification. The last step was to determine whether the recombinantly produced cationic peptides were biologically active.  M I C tests were done on these partially purified  peptides (Table V ) . The results showed that all three cationic peptides had activity against Gram-negative E. coli and P. aeruginosa and no activity against Gram-positive bacteria. Both bands of bactenecin had moderate activity against the wild type Gram-  38  1  2  3  4  Figure 4: Cleavage of cationic peptides from protein A. Cationic peptides were cleaved from protein A by CNBr (described in Material and Methods). After the digestion, samples were run on an A U gel. Lanes: 1, synthetic C E M E as control; 2, bactenecin (upper band is multimer, lower band is the intra-chain, disulphidedoonded product); 3, apidaecin; 4, indolicidin. The arrows indicate cationic peptide bands.  39  0.14  0.12  0.08 o  oo CN  0.06 -  0.04 A  0.02  11  16  21  26  31  •Fraction Number  Figure 5A: Elution profile of the CNBr-digested protein A/apidaecin fusion from a BiogelPlOO Column. After C N B r digestion, the sample was diluted 100 times with distilled water and lyophilized.  The dried pellet was dissolved in 1 % acetic acid and applied to a Biogel  PI00 column. Peptides were eluted with 1 % acetic acid.  40  Figure 5 B : Purification of apidaecin on a Biogel PI00 column. One mililitre fractions were collected.  Five microlitre samples from every second  fractions (Fig 5A) were run by acid-urea gel electrophoresis.  Lanes: 1, C N B r digested  mixture as control; 2-9, fraction 8, 10, 12, 14, 16, 18, 20, 22.  The arrows indicate  apidaecin bands.  41  0.35  Fraction Number  6 A : Elution profile of the CNBr-digested protein A/bactenecin fusion from a Biogel PI00 Column.  Figure  After CNBr digestion, the sample was diluted 100 times with distilled water and lyophilized. The dried pellet was resuspended in 1 % acetic acid and applied to a Biogel PI00 column. Peptides were eluted with 1 % acetic acid.  42  *0- i  .**  Figure 6B: Purification of bactenecin on a Biogel PI 00 Column. One mililitre fractions were collected. Five microlitre samples from each peak (Fig 6A) were run by acid-urea gel electrophoresis. Lanes: 1, control; 2-10, fractions 7, 11, 25, 26, 27, 28, 29, 30, 31. The arrows indicate both the upper band (multimer) and lower band of bactenecin (intra-disulphide-bridged monomer).  43  Figure 7 A : Elution profile of the CNBr-digested protein A/indolicidin fusion from a Biogel PI00 Column. After CNBr digestion, the sample was diluted 100 times with distilled water and lyophilized. The dried pellet was resuspended in 1% acetic acid and applied to a Biogel PI00 column. Peptides were eluted with 1% acetic acid.  44  1  2  3  4  5  6  7 8  9  10  9m  11 12 13  14  15  16  17  18  19  20  21  Figure 7B: Purification of indolicidin on a Biogel PI00 Column. One mililitre fractions were collected.  Five microlitre samples from every third fraction  (Fig 7A) were run by acid-urea gel electrophoresis.  Lanes: 1, control; 2-10, fraction 9,  12, 15, 18, 21, 25, 28, 31, 34; 11-20, fraction 44-54. The arrows indicate the indolicidin bands.  45  Table V : MICs of Three Partially Purified Cationic Peptides Expressed in S. aureus MIC pg/ml Apidaecin Species  Strains  E.coli  UB1005 DC2 K799 Z61 K147 SAP0017  P. aeruginosa S. aureus S.epidermidis B. subtilis  Indolicidin  Bactenecin Lower Band Upper Band  8 1 64 32 >64 >64 >64 >64  32 4 >64 n/a >64 >64 >64 >64  32 8 16 4 >64 >64 >64 >64  64 4 32 2 >64 >64 16 >64  negative bacteria E. coli (32 pg/ml) and P. aeruginosa (16-32 pg/ml). Indolicidin was active against E. coli and its mutant. Apidaecin had the lowest M I C (8 pg/ml) of the three peptides against E. coli. None showed any activity against Gram-positive bacteria.  C. Production of Bactenecin in E. coli While the fusion of cationic peptides to protein A proved to be an effective and convenient way to produce pure cationic peptides, there are two major drawbacks to this system: First, S. aureus is a pathogen and is not an industrial standard strain; secondly, the IgG column matrix is expensive. Another alternative would be to utilize E. coli as expression vector and a less expensive cellulose-binding domain as affinity tag. Plasmid pSP72 contained a fusion partner including Rep 21 (a truncated version of Rep A), a synthetic cellulose-binding domain and a hexa histidine domain as well as the prepro region of human defensin 1 (ELNP-1) (Zhang et al., 1998). To transfer the  46  cationic peptide gene from plasmid pRIT5 to pSP72, a primer (primer 3, F i g . 2C) was designed to engineer the EcoRI site to an E.coRV site at the 5'-end.  Another primer  (primer 2, F i g . 2C), downstream of the polylinker at pRIT5 was also used in the P C R amplification to obtain the fragment of cationic peptide gene from pRIT5.  These  fragments were digested with appropriate restriction enzymes, and cloned into pSP72. E. coli B L 2 1 harboring the pSP72/bactenecin construct was grown in L B medium with 100 pg/ml ampicillin until the cell density reached an O D  6 0 0  of 0.4, then was induced with 1  m M I P T G and grown for an extra 3-4 hours before harvesting.  The expression of  bactenecin fusion protein was detected (Fig. 8). With vector alone, lanes 1 and 2 showed the exact same protein patterns with or without I P T G induction.  With the bactenecin  construct, lane 4 showed extra band (as expected at 15.2 K D a , arrow indicated) with I P T G induction, which wasn't present without I P T G induction (lane 3).  Attempts to  extract inclusion bodies were made, but insufficient fusion protein was obtained to do cleavage and M I C tests. The E. coli expression system proved to produce significantly more peptide fusion protein than the protein A system (Zhang et al., 1998). While efforts are being made to improve the bacterial expression system in our laboratory, including utilizing a high expression vector pT7-7 (Zhang et al., 1998), the focus of the thesis shifted to the antimicrobial mechanism of bactenecin.  47  1  2  3  5  4  Figure 8: Expression of bactenecin in E. coli. E. coli harboring plasmid pSP72/bactenecin was grown until OD oo 0.6-l, and induced by 1 m M IPTG. After the induction, the culture was grown for additional 3-4 hr. Samples of whole cell lysates were run on SDS-PAGE. Lanes: 1, molecular weight markers; 2, E. coli pSP72 without IPTG induction; 3, E. coli pSP72 with IPTG induction; 4, E. coli pSP72/bactenecin without IPTG induction; 4, E. coli pSP72 with IPTG induction. The arrow indicates the fusion protein. =  6  48  D. Summary  Cationic  peptides  apidaecin, indolicidin  and  bactenecin  were  produced  recombinantly and proved to be active against the Gram-negatives E. coli and P. aeruginosa. Prior to this study, only longer (26-28 amino acid) a-helical peptides had been recombinantly produced as fusion proteins, and this study demonstrated the capability of the system for expressing shorter peptides (12-18 amino acids) including one with a disulphide bridge (bactenecin) and two with unusual compositions (indolicidin with >40% tryptophan, and apidaecin with 5 proline residues). Having proven that these peptides could be recombinantly expressed, it was decided to concentrate on one of these shorter peptides bactenecin, and study its mechanisms of its action.  49  CHAPTER TWO: ANTIBACTERIAL M E C H A N I S M OF BACTENECIN  A . Introduction Bactenecin was previously found to be active against E. coli and S. aureus (Romeo, et al., 1988), and strongly cytotoxic  for rat embryonic neurons, fetal rat  astrocytes and human glioblastoma (Radermacher, et a l , 1993). However, little is known about its antimicrobial mechanism and whether it shares a common killing mechanism with many other antimicrobial peptides by perturbing the bacterial membrane, or i f it has a distinct mode of action due to its unique compact structure (c.f. the silk moth peptide cecropin which is a 26-amino acid amphipathic a-helix).  Its small size and single  disulphide bond also makes bactenecin an interesting candidate for research and drug development.  The aim o f this chapter was to investigate how bactenecin interacts with  and kills microoganisms. The amino acid sequence of bactenecin and its linear derivative are shown in Fig. 9.  The linear derivative (bac2S) with two cysteine residues replaced by two serine  residues, was made to determine the importance of the disulphide bond in bactenecin's antimicrobial activity. The reduced form of bactenecin was also included in this study as a linear version of bactenecin. The identity of these peptides was confirmed by M A L D I mass spectrometry.  The M A L D I data showed the molecular weight of the reduced  bactenecin as 1486+1 dalton and oxidized bactenecin as 1484+1 dalton, in agreement with formation of one disulphide bond in the latter.  50  Bactenecin: R L C R I V V I R V C R Lin-Bac: Bac2S:  Figure 9:  RLCRIVVIRVCR RLSRIVVIRVSR  Amino acid sequence of bactenecin and its linear variants.  The cysteine residues that are involved in the disulphide bond formation are underlined.  51  B. A n t i m i c r o b i a l Activity The M I C s of bactenecin and its derivatives against a range o f bacteria was determined (Table VI) by using a modified broth dilution method. Bactenecin was active against all Gram-negative bacteria tested.  It was relatively inactive (MIC=64 pg/ml)  against the Gram-positive bacterium S. aureus, in contrast to a previous report (Remeo, et al., 1988). The linear variant bac2S and reduced bactenecin were inactive against wild type Gram negative bacteria. P. aeruginosa Z61 and E. coli D C 2 are outer-membranebarrier-defective mutants, that have more permeable outer membranes than their parent strains, allowing potentially easier access of the peptides to the cytoplasmic membrane. A l l three bactenecins exhibited equivalent activity against these two mutants. bactenecin  and bac2S  showed  somewhat  improved activity  against  the  Native defensin  supersusceptible phoP/phoQ mutant of S. typhimurium, while reduced bactenecin was inactive against this mutant. Linearization by changing cysteine to serine or by reduction dramatically changed the antimicrobial activity against two Gram-positive species. The linear bactenecins were active against S. epidermidis and E. facaelis, while the native bactenecin was inactive against S. epidermidis and E. facaelis.  Bac2S also showed  moderate activity (16 pg/ml) against S. aureus. In order to see i f linear bactenecins are in general more Gram positive specific, six other Gram positive strains were tested. Linear bactenecins were 8x and 2-4x more active against L. monocytogenes and S. mitis respectively than was the native bactenecin.  Reduced bactenecin was active against S.  pneumoniae, while both bac2S and native bactenecin were relatively inactive against S.  52  CD O  S3 T3 CD  rt  NO  A  o  CN  hi"  hi" NO 00 NO  NO  A  A  A  oo  IT)  NO NO  A  A  ho  NO  o  CD  h*  ho  NO  A  cCD  *  o rt  6J) 00 CN  CN  O  rt cn  O  rt CQ  CN  u PQ  CN  hi" NO  hi" cn NO NO  A  A  hf  A  NO  NO  NO  NO  NO  A  in ©  1  PI • i-H  T3  o  PL,  -a <D O  CD N  'HI Q.  NO CN  S3 CD  rt  o  T3  O  00 CN O  ca  PQ  > CD  p  ©  i-H I  oo  hi" oo NO  A  NO  A  NO  A  _  (D >  CD ft +-»  CN  O c Q CD o .fi O  I>  CD >  00 I 00 »—H  CO  w  Pi CD  PI  CD  CD  CD 'CD  O  CO  3  CD  CD CD  ft  "rt o  i<8  rt PQ o co  u  > — 3  ON ON  u f-  +-»  Q  00  «  ON NO ON  O H  O u U H H  © U © 00  in NO U o  00  CXI OO  s  CD  •3  I*  cj  N  1^  cu  <3  Cd  cu  ,  •2  * o R  s  •§>  o  ^  CO.  as  <  R  53 ,  NO  in  cu  'o  s  •a  CO  CD  CD  CD  <D  CD  CO  CN  ft l-q  co  o  CN  fi  ft  'o  PI  CL  rt  CD  co  CO  o  PL,  CD CD  ON  O  A  CD,  o  CD '  CN  rt  CN  PI  NO  CD fi <D »  cn  <D  h* NO  A  ICO  cs CJ cu  CO  S  CU i<  3  I CO  O  to  O  cu  R  5  o  u £ < s R  s  R  to  CL  cu  CO 03  -a o H _c  CD  P  CD T3 CD sCD CO  CD  *rt >  pneumoniae.  C. Secondary Structure by Circular Dichroism Circular dichroism (CD) spectrometry (Fig 10A) showed that bactenecin in the reduced form and bac2S were present in 10 m M sodium phosphate buffer as unordered structures which had strong negative ellipticities near 200 nm. Native bactenecin had a spectrum resembling oxyribonuclease and nuclease that are short polypeptides, each with a disulphide bond (Venyaminov et al., 1996). The C D spectrum o f bactenecin (Fig 10A), demonstrated a negative ellipticity near 205 nm, typical o f that seen for a type I B-turn structure (Perczel et al., 1996). In 60 % T F E buffer, in the presence of liposomes and 10 mM  S D S , the native bactenecin retained a similar structure (Fig 10B, 10C and 10D).  However, in that medium, the reduced form and bac2S exhibited clearly distinct structures from those observed in aqueous solution.  In 60% T F E buffer (considered a  helix-inducing solvent), these two peptides tended to form a-helical structures (Fig 10B), whereas in the presence of liposomes or l O m M S D S , p-sheet structures were evident (Fig.  10C, 10D). S D S micelles resemble the environment of lipid membranes.  D. Interaction with Bacterial cells 1. Interaction with outer membrane of Gram negative cells 1) The binding ofbactenecins  to purified LPS from E. coli UB1005  54  4  3  2  Figure 10A: C D spectra of bactenecin, its reduced form and bac2S in phosphate buffer. The concentrations o f peptides were 50 p M . C D measurements were taken in 10 m M sodium phosphate buffer (pH 7.0). Bactenecin:  ; Reduced bactenecin: — ; Bac2S: -  55  Wavelength (nm)  Figure 10B: C D spectra of bactenecin, its reduced form and bac2S in 60% (v/v) T F E . The concentrations of peptides were 50 p M . Bac2S: - - -.  Bactenecin:  ; Reduced bactenecin:  Wavelength (nm) Figure IOC: C D spectra of bactenecin, its reduced form and bac2S in 60% (v/v) T F E . The concentrations of peptides were 50 p M . Bactenecin: Bac2S: - -.  ; Reduced bactenecin:  4  .4 '  Wavelength (nm)  Figure 10D: C D spectra of bactenecin, its reduced form and bac2S in l O m M S D S . The concentrations of peptides were 50 p M . Bactenecin: Bac2S: - -.  ; Reduced bactenecin: — ;  58  The  M I C results indicated that the interaction o f bactenecin with the outer  membrane might be critical in causing the differences in antimicrobial activity against Gram negative bacteria among the three bactenecin forms.  The first step of cationic  peptide antimicrobial action involves the binding o f the cationic peptide to the negatively charged surface o f the target cells (Hancock et al., 1995). In Gram negative bacteria, this initial interaction occurs between the cationic peptides and the negatively charged L P S in the outer membrane (Falla, 1996; Sawyer, 1988; Piers, 1994). quantified using the dansyl polymyxin B displacement assay.  Such binding can be  Dansyl polymyxin B is a  fluorescently tagged cationic lipopeptide, which is non-fluorescent in free solution, but fluoresces strongly when it binds to L P S .  When the peptides bind to L P S , they displace  dansyl polymyxin B resulting in decreased fluorescence, which can be assessed as a function of peptide concentration (Fig. 11). Bactenecin was a relatively weak LPS-binder compared to polymyxin B , but it was still better than M g , the native divalent cation 2 +  associated with L P S .  Most importantly, it seemed that native bactenecin bound to L P S  far better than its linear derivative and reduced form, which may partially explain the difference in their activities against Gram negative bacteria.  2) Effect on outer membrane permeability Antimicrobial peptides bind to L P S , displacing bound divalent cations.  Due to  their bulky nature they disrupt the outer membrane and promote their own uptake across the outer membrane (Sawyer, 1988; Piers, 1994).  In order to determine whether better  binding ability resulted in better outer membrane permeabilization, an N P N assay was  59  Figure 11: Binding of peptides to LPS as assessed by their ability to displace dansyl polymyxin B from E. coli UB1005 LPS.  Dansylpolymyxin B was added to 1ml of 3 pg/ml LPS to a final concentration of l p M which saturated the binding sites on LPS, and the fluorescence sensitivity was adjusted to 90%. The peptides and M g were titrated in resulting in a decrease in fluorescence due to dansyl polymyxin displacement until no decrease or very small decrease was detected. Polymyxin B: - X - ; M g C l : Bactenecin: - A - ; Bac2S ^ B - ; Bactenecin (reduced): 2+  2  60  100  0.1  1  10  100  Concentration (ug/ml)  Figure 12: Peptide-induced outer membrane permeabilization measured by the N P N assay uptake in E.  coli UB1005.  Mid-log phase E . coli cells were collected and incubated with N P N in the presence of various concentrations of bactenecin (oxidized), bac2S, or bactenecin (reduced). was taken up into cells when the outer membrane was disrupted by the peptides. uptake of N P N was measured by an increase in fluorescence.  NPN The  Polymyxin B :  Bactenecin: -+-; Bac2S: -o-; Bactenecin (Reduced): - V -  61  performed.  N P N is a neutral hydrophobic probe that is excluded by an intact outer  membrane, but is taken up into the membrane interior of an outer membrane that is disrupted by antimicrobial peptide action. strongly when it enters the membrane.  N P N fluoresces weakly in free solution but  F i g . 12 shows that polymyxin B permeabilized  the outer membrane to 50% of maximal increase in fluorescence arbitrary units at 0.4 pg/ml,  while  bactenecin,  bac2S  and  linear  bactenecin  caused  permeabilization at 0.8 pg/ml, 2 pg/ml and 4.5 pg/ml respectively.  half  maximal  Native bactenecin  thus appeared to be better than the linearized derivative and its reduced form at permeabilizing the outer membrane of E. coli UB1005.  E. Summary Bactenecin was a type I P-turn molecule either in an aqueous or in a membranelike environment. It was active against the Gram-negative bacteria E. coli, P. aeruginosa, and S. typhimurium,  but was relatively inactive, or less active, against Gram-positive  bacteria compared to its linear and reduced derivatives.  Bactenecin bound to negatively  charged L P S from the E. coli outer membrane and permeabilized the outer membrane to allow the small hydrophilic N P N molecules into cells.  However bactenecin did not  appear to be a strong outer membrane permeabilizer compared to polymyxin B . Bactenecin appeared to bind to L P S and permeabilize the outer membrane better than its linear and reduced derivatives, which explained its better antimicrobial activity against Gram-negative bacteria.  The disulphide bond seemed to be critical for bactenecin to  interact with the outer membrane of Gram-negative bacteria.  62  Although bactenecin interacted with the outer membrane of bacteria by the same mechanism as typical peptides (e.g. defensin, cecropin), how it interacted with and eventually  killed bacteria is still unclear.  Therefore, Chapter Three focuses  on  developing a cytoplasmic membrane permeability assay to study the interaction with the cytoplasmic membrane.  63  C H A P T E R T H R E E : INNER M E M B R A N E P E R M E A B I L I T Y  A . Introduction It has been proposed that the antibacterial target of cationic peptides is at the cytoplasmic membrane (Duclohier et al., 1989). Cationic peptides are generally able to interact  electrostatically  with  the  negatively  charged  headgroups  of  bacterial  phospholipids and then insert into the cytoplasmic membrane, forming channels or pores which are proposed to lead to the leakage of cell contents and cell death (Christensen et al., 1988; Lehrer, 1989; Wimley, et al., 1994).  However there are very few data for  peptides pertaining to measurement of the disruption of the cytoplasmic membrane permeability barrier, despite ample evidence that membrane disruption can occur in model membrane systems (Silvestro et al., 1997). Although some authors have utilized measurements of the accessibility of a normally-membrane-impermeable substrate to cytoplasmic B-galactosidase, such assays suffer from using a bulky substrate (ortho nitrophenyl galactoside)  (Skerlavaj, 1990; Lehrer, 1989), as well as an inability to  dissociate inner membrane from outer membrane permeabilization (Lehrer, 1989).  To  circumvent this I have adopted an assay involving the membrane potential sensitive dye diSC3-(5) to measure the disruption of the electrical potential gradients across the cytoplasmic membrane in intact bacteria. Bacteria, through either electron transport or A T P hydrolysis, eject protons and maintain an energized membrane or proton motive force comprising an electrical potential gradient (A\\i, oriented internal negative) and a p H gradient (ApH, oriented internal alkaline) (Harold, 1972). Since the cytoplasmic p H of E.  64  coli is maintained at a p H 7.4, at neutral p H in the external medium, by far the major contribution to the protonmotive force is provided by the membrane potential gradient of about -150 m V (Bakker, 1978). The homeostasis between A p H and A\\i is maintained by potassium flux utilizing the large potassium pool inside cells (approximate concentration 100-150 m M K ) . Permeabilization of the cytoplasmic membrane would allow ions to +  equilibrate across the cytoplasmic membrane to reduce or destroy the membrane potential gradient. The use of E. coli mutant D C 2 allowed me to perform this assay in the absence of E D T A [required by previous workers who have used similar assays in E. coli (Letellier and  Shechter, 1979; Ghazi et al., 1981)]. E. coli D C 2 is an outer membrane defective  mutant, which has an outer membrane that is more permeable to hydrophobic, small hydrophilic and polycationic peptide molecules (Richmond, et al., 1976). The fluorescent probe diSC3-(5), which is a caged cation (a positively charged quaternary ammonium buried within a hydrophobic matrix), distributes between cells and the medium depending on the cytoplasmic membrane potential gradient.  Once it is inside the cells, it becomes  concentrated and self-quenches its own fluorescence (Sim, et al., 1974). The quenching is probably due to the formation of dye aggregates inside cells with reduced fluorescence (Sim,  et al., 1974).  If peptides form channels or otherwise disrupt the membrane, the  membrane potential will be dissipated, and diSC3-(5) will be released into the medium causing the fluorescence to increase, as can be detected by fluorescence spectrometry. In these assays, 0 . 1 M K C I (final concentration) was added to the buffer to balance the chemical potential of K  +  inside and outside the cells.  65  B. Fluorescence Quenching of diSC3-(5) by E. coli DC2 cells Fig.  13 demonstrates the fluorescence changes of the diSC3-(5) upon addition to  mid-log phase E. coli D C 2 cells (OD =0.05) at room temperature. The fluorescence 600  decreased gradually until it stabilized to a steady level after an hour. The fluorescence quenching was induced in E. coli cells with an intact membrane potential gradient (inside negative).  The addition of 0.1M KC1 without the presence of valinomycin after the  stabilization had no effect on the fluorescence intensity.  C. The relationship of K concentration outside the cells and the fluorescence +  intensity in the presence of valinomycin Changing the concentration o f potassium outside the cells will change potassium concentration gradient accordingly.  the  The potassium concentration gradient  opposes the electrical gradient and thus maintains the membrane potential gradient A\p across the membrane (Letellier and Shechter, 1979). Valinomycin, an ionophore renders the cytoplasmic membrane permeable to potassium (Harold, 1970). Therefore varying the K  +  concentration in the presence of valinomycin outside cells will change the equilibrium  Avj/ accordingly. This was indicated by plotting the fluorescence quenching of diSC3-(5) as a function o f K concentration outside the cells (Fig. 14). +  A s the concentration of K  outside increased, the fluorescence intensity increased proportionally (ie.  +  fluorescence  quenching decreased as diSC3-5 left the cells) indicating a decrease o f membrane potential. A linear relationship was observed over the range of 10 m M to 100 m M  66  0  10  20  30  40  50  60  Time (minutes)  Figure 13: Fluorescence quenching of diSC3-(5) by log-phase E. coli cells. DiSC3-(5) at 0.4 p M was added to log-phase E. coli cells ( O D  6 0 0  = 0.05) resuspended in  5 m M H E P E S and 5 m M glucose (pH 7.2), and incubated at room temperature.  67  100 90 -  External K C I concentration ( m M ) Figure 14: Effect of external KCI concentration on the fluorescence intensity of diSC3(5) incubated with E. coli D C 2 cells and valinomycin. Log-phase E. coli cells (OD oo = 0.05) were incubated with 0.4 p M diSC3-(5) until 6  maximal fluorescence quenching was observed and then 1 p M valinomycin was added. Various concentrations of KCI were added to 1 ml aliquots of the mixture to alter the K  +  gradient and were incubated at room temperature for 10 min before fluorescence readings were taken.  68  external potassium concentrations.  A t 0.1M KC1, the increase in fluorescent intensity  reached its maximum, due to equilibration of the outside and inside K  +  concentrations.  This result proved that the partitioning of diSC3-(5) in the suspension medium and inside cells was membrane potential - dependent and was directly proportional to the size of the membrane potential.  T o prevent any effects due to the movement of potassium (which  could oppose the dissipation of the electrical potential gradient), an external concentration of 0.1 M KC1 was used in the subsequent cytoplasmic membrane permeability assay. A s a control, it was shown that 1 p M valinomycin alone did not cause a change in fluorescence intensity, while subsequent addition of 0.1 M KC1 resulted in a very rapid immediate release of diSC3-(5) (Fig. 15). Cationic peptides have been hypothesized to form pores or channels in the membrane and cause the cell to leak out their cytoplasmic contents.  When such pores or  channels are formed, membrane potential will be dissipated and diSC3-(5) would be redistributed accordingly, causing the fluorescence to increase. permeability assay was developed based on this assumption.  The inner membrane  Gramicidin S (16 pg/ml)  increased the fluorescence intensity by about 30 arbitrary units, while subsequent addition of 0.1 M KC1 resulted in another 30 unit increase (Fig 16). Presumably the first increase in fluorescence  was due to redistribution of both protons and K  +  to establish the  membrane potential at a new (lower) level, whereas in the presence of 0.1 M K which +  equilibrated the transmembrane K  +  concentration gradient, the membrane potential was  completely dissipated.  69  Figure 15: Kinetics of fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 cells in the presence of valinomycin and K C I . Log-phase  E.  coli cells were incubated with  0.4  p M diSC3-(5)  until maximal  fluorescence quenching was obtained. Valinomycin l p M and 0.1M K C I were added to a 1 ml aliquot when indicated by arrows.  70  Figure 16: The effect of 0.1 M KC1 on the dissipation of cytoplasmic membrane potential caused by gramicidin S. Gramicidin S (16 pg/ml) was added to log-phase E. coli cells that had been preincubated with 0.4 p M diSC3-(5), and an increase of fluorescence intensity was observed.  When  the fluorescence increase was stable, 0.1M KC1 was added which resulted in an additional fluorescence increase.  71  D . Antimicrobial activities in the presence of potassium chloride Since 0 . 1 M K C I was added to the assay buffer to equilibrate the cytoplasmic and external K concentration, the effect of 0.1 M K C I on the antimicrobial activity was +  tested.  Table VII shows the minimum inhibitory concentrations (MIC) of a variety of  cationic peptides in the presence and absence of K . It seemed that the activity of the cc+  helical cationic peptides was not affected by the presence of K . A m o n g the (3-structured +  peptides tested, the analog Gram 474 was not affected, the M I C s of gramicidin S and Gram 4112 were increased by 4 and 8 fold respectively.  The M I C s of the extended  structured peptides C P 1 0 C N and its variant C P 1 1 C N were increased by 2 to 4 fold. The M I C of polymyxin B did not change, but M I C s of bactenecin, its reduced form and bac2S were increased by 4 to 8 fold.  E. The interaction of a-helical cationic peptides with the cytoplasmic membrane CP26, CP27, CP28 and CP29 belong to the group of amphipathic a-helical peptides (Table IV). CP27 ( C E M E , MBI-27) is a hybrid of cecropin (residues 1-8) and mellitin (residues 1-18) (Wade, et al., 1992). CP28 ( C E M A , MBI-28) is a derivative of CP27 with two extra positive charges at the C-terminal end (Piers, 1994).  CP26 and  CP29 were also CP27 derivatives designed in our laboratory by Dr. Nedra Karunaratne to have improved a-helicity. Although these four peptides had only 2-4 fold differences in M I C s , their abilities to dissipate the membrane potential were quite different (Fig 17). CP26 had the lowest  72  T a b l e V I I : M I C s of E.coli D C 2 in the Presence and Absence o f K  M I C tig/ml Peptide  K+=0  K+=0.1M  CP26  0.25  0.5  CP27  2  2  CP28  0.5  1  CP29  1  1  2  8  32  32  1  8  CP 10CN  4  16  CP 11CN  2  4  0.0156  0.0156  Bac 2S  2  16  Bactenecin (reduced)  2  16  Bactenecin  2  8  a-helical  ^-structured Gramicidin S Gramicidin 474 Gramicidin 4112  extended structured  Polymyxin B  loop-structured  +  100  80 A  -20 J  Time (minutes)  Figure 17: Kinetics o f fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 in the presence of a-helical cationic peptides. a-Helical peptides at their M I C s were added seperately to respective log-phase E. coli D C 2 cells preincubated with 0.4 p M diSC3-(5) in the presence of 0.1 M KC1. fluorescence changes with time were recorded. CP26: 0.5 pg/ml, •-;  The  CP27: 2 pg/ml, -  CP28: 1 pg/ml, - A - ; CP29: 1 pg/ml, - X - .  74  MIC,  yet it failed to dissipate the membrane potential at its M I C .  It started to disrupt the  membrane potential at 1 pg/ml, two fold higher than its M I C and achieved maximal membrane potential disruption at 8 pg/ml (Fig 18).  CP27, CP28 and CP29 had similar  M I C s and they showed a similar pattern (Fig 17 and 18) in completely dissipating the membrane potential at concentrations within two-fold of their M I C s . These fluorescence increases caused by CP27, 28, 29 stabilized within 2 min. CP27 had the highest M I C among these peptides, but it seemed to have the greatest ability to dissipate the membrane potential.  F. The interaction of P-structured cationic peptides with the cytoplasmic membrane Gramicidin S is a naturally occurring cyclic peptide antibiotic first isolated from Bacillus brevis (Gause and Brazhnikova, 1944).  It has been shown to possess a two-  stranded P-sheet structure connected by P-turns (Izumiya, et al., 1979; Rackovsky and Scheraga, 1980). Gram 474 and Gram 4112 are two analogs of gramicidin S synthesized by  Dr. Kondajewski and Dr. Hodges in collaboration with Dr. Hancock (Table IV).  Gram S and Gram 4112 had similar antimicrobial activity against E. coli, while Gram 474 was 4 times less active (Table VII). At  their  M I C , all  depolarization (Fig. 19),  three  gramicidin peptides  although Gram 4112  membrane potential as the other two.  caused  rapid  membrane  was not as effective in dissipating  Both gramicidin S and Gram 474 started to cause  membrane permeabilization at very low (sub-MIC) concentrations and achieved maximal dissipation of membrane potential at concentrations well below their M I C s , at 2 pg/ml  75  90 -  Figure 18: Effect of a-helical cationic peptides on the fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 cells. Various concentrations of cationic peptides were added to 1 m l log-phase E. coli D C 2 cells that had been pre-incubated with 0.4 p M diSC3-(5) in the presence of 0.1 M  KC1.  Readings were taken when the maximal fluorescence increase was reached typically within 5 min.  CP26:  CP27:  CP28: - A - ; CP29: - X - . Arrows indicated M I C  values.  76  T i m e (minutes)  Figure 19: Kinetics of the fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 in the presence of p-structured cationic peptides. P-Structured peptides at their M I C s were added to log-phase E. coli D C 2 cells that had been pre-incubated with 0.4  p M diSC3-(5) in the presence of 0.1  fluorescence changes with time were recorded. Gramicidin S: 8 pg/ml,  M KCI.  The  G474: 32  p g / m l , G 4 1 1 2 : 8 pg/ml, - A - .  77  (Fig.  20).  Gram 4112  on the other hand, did not cause maximum membrane  permeabilization at its M I C , and achieved this only at >40 pg/ml (ie. >5x M I C ) .  G. The interaction of extended structured cationic peptides with the cytoplasmic membrane Peptide C P 1 0 C N , with its carboxyl terminus amidated, is identical to the 13amino acid antimicrobial peptide indolicidin, present in the cytoplasmic granules of bovine neutrophils (Selsted, et al., 1992). C P 1 1 C N is a derivative o f C P 1 0 C N with Pro3 and Trp4 substituted with Lys3, and an additional A r g residue at the C-terminus producing a molecule with a greater positive charge (Falla and Hancock, 1997). At their M I C s , both peptides increased membrane permeability rapidly (Fig. 21). However the increase was only 30-35 arbitrary units (cf. CP27). These peptides failed to cause full dissipation o f cytoplasmic membrane potential even at concentrations 4 fold the M I C (Fig. 22) and quite different concentration dependences were observed.  H. The interaction of looped structured cationic peptides with the cytoplasmic membrane The M I C s of bactenecin, reduced bactenecin and bac2S in the presence of 0.1 M KC1 were determined and shown to be 8-16 pg/ml (Table VII).  Despite these similar  M I C s for the 3 peptides against E. coli D C 2 , the influence of different concentrations of these peptides on the membrane potential was quite different (Fig. 23 and 24). The native bactenecin started to cause the release of probe at half-MIC concentrations, whereas its  78  100  90  H  0  H  0.1  ,  1  ,  1 10 Peptide concentration (mg/ml)  100  Figure 20: Effect of p-structured cationic peptides on fluorescence intensity of diSC3(5) incubated with E. coli D C 2 cells. Various concentrations of cationic peptides were added to 1 ml log-phased E. coli D C 2 cells incubated with 0.4 p M diSC3-(5) in the presence of 0.1 M K C 1 .  Readings were  taken when the maximal fluorescence increase was reached. Gramicidin S: ^ ; Gram474: Gram 4112: - — - . Arrows indicated the MICs.  79  100  T i m e (minutes) Figure 21: Kinetics of fluorescence intensity changes o f diSC3-(5) incubated with E. coli D C 2 in the presence of extended cationic peptides. Extended peptides at their M I C s were added to log-phase E. coli D C 2 cells incubated with 0.4 p M diSC3-(5) in the presence of 0.1 M KC1. The fluorescence changes with time were recorded. C P 1 0 C N : 8 pg/ml,  C P 1 1 C N : 4 pg/ml,  CP27: 2 pg/ml, - A - .  80  Peptide concentration (mg/ml)  Figure 22: Effect of extended cationic peptides on the fluorescence intensity of diSC3(5) incubated with E. coli D C 2 cells. Various concentrations of cationic peptides were added to 1 ml log-phase  E. coli D C 2  cells that had been pre-incubated with 0.4 p M diSC3-(5). Readings were taken when the maximal fluorescence increase was reached.  CP10CN:  CP11CN:  Arrows  indicate the M I C .  81  T i m e (minutes)  Figure 23: Kinetics of fluorescence intensity changes of diSC3-(5) incubated with E. coli D C 2 in the presence of loop-structured cationic peptides. Loop-structured peptides at their M I C s were added to log-phase E. coli D C 2 cells that had  been pre-incubated with 0.4 p M diSC3-(5) in the presence of 0.1 M KC1. The  fluorescence changes with time were recorded. (reduced): 16 pg/ml,  Bactenecin: 8 pg/ml,  Bactenecin  Bac 2S: 16 pg/ml, - - . A  82  Figure 24: Effect of loop-structured cationic peptides on the fluorescence intensity of diSC3-(5) incubated with E. coli D C 2 cells. Various concentrations of cationic peptides were added to 1 m l log-phase E. coli D C 2 cells that had been pre-incubated with 0.4 u M diSC3-(5) in the presence of 0.1 M  KCI.  Readings were taken when the maximal fluorescence increase was achieved. Bactenecin: -—-; Bactenecin (reduced):  Bac 2S:  Arrows indicate the M I C s .  83  linear  derivative  bac2S  and reduced bactenecin  showed  maximal  probe  release  (dissipation of membrane potential) at concentrations well below the M I C concentration (Fig. 24). Bactenecin caused the permeabilization gradually and very slowly, and even at 64 pg/ml it reached maximum permeabilization at 90 minutes, while reduced bactenecin reached its maximal permeabilizing effect within 4 min (Fig. 23). somewhat like reduced bactenecin.  Bac 2S behaved  These results agreed with the kinetics observed in a  killing assay.  G. Summary A n assay was developed to study the interaction of cationic peptides with the cytoplasmic membrane and the importance of transmembrane potential.  It seemed that  different peptides behaved quite differently to depolarize the membrane potential. There was no consistent correlation between the ability to permeabilize the  cytoplasmic  membrane and the antimicrobial activity.  84  CHAPTER FOUR: STRUCTURE AND FUNCTION RELATIONSHIPS  A. Introduction Cationic peptides share common features of being highly cationic and forming amphipathic structures.  Their cationic feature facilitates their interaction with the  negatively charged cell surface and their amphipathic structures enable them to be incorporated into the hydrophobic membrane interior to form channels and pores which have been proposed to ultimately cause cell death. However, bactenecin was showed to function in fundamentally different way to its linear variants and other known cationic peptides, like defensin and cecropin, due to its unique structure. Its basicity still allowed it to interact with the negatively charged outer membrane and be translocated across the outer membrane by the self-promoted uptake pathway, the same mechanism as proposed for  a-helical and P-structure peptides.  However, it presumably used a different  mechanism to interact with and translocate across the cytoplasmic membrane, and it may attack an unknown cellular target leading to cell death. The linear variants of bactenecin created either by reduction (Lin-Bac) or serine substitution (Bac2S) lost the ability to interact with the negatively charged outer membrane due to the disruption of the disulphide bond, but were able to interact with the cytoplasmic membrane, possibly by forming channels and pores (although multimers or aggregates have to be involved). The different secondary structure that they adopted when came in contact with the membranelike environment (e.g. liposome) may account for the difference in the mechanism.  85  The  structural  features  (cationicity,  disulphide  bond,  amphipathicity)  of  bactenecin and its linear variants seemed to play a key role in the antimicrobial mechanism.  Therefore, work in this chapter was designed to study structure:function  relationship.  Hydrophobicity, positive charges, disulphide-bridging and amphipathicity  are also recognized as important factors in the antimicrobial activities o f cationic peptides.  Analogues were designed to investigate the.effects o f modification of these  factors on antimicrobial activities.  These analogues can be divided into three groups,  linearized, more positive in charged, and increased hydrophobicity of the ring.  B. Linearization In Chapter I, two linear derivatives o f bactenecin, bac2S and reduced bactenecin were described.  They were found to have higher selectivity against Gram-positive  bacteria, and no activity against wild type Gram-negative bacteria. T o further confirm this observation, two more linear derivatives of bactenecin were made, b a c 2 A - C N (with two cys to ala replacements) and b a c S - C N (with a single cys-3 to ser-3 replacement). The hydroxyl groups in serine residues and the sulphydral groups in cysteine residues are capable of hydrogen bonding to water and thus are hydrophilic, and would tend to make linear bactenecin more hydrophilic than cyclic bactenecin since the S H groups in native bactenecin are joined to form a disulphide bridge.  T o maintain the hydrophobicity of  linear and native bactenecin as similarly as possible, b a c 2 A - C N had alanine substitutions at both cysteine positions, since alanines are hydrophobic residues. Both b a c 2 A - C N and b a c S - C N were amidated at the carboxyl terminal end. Both amidated and non-amidated versions of bac2S were also included in this study.  86  A l l three peptides were found to be more active against Gram-positive bacteria compared to native bactenecin (Table VIII). While native bactenecin was inactive against S. aureus, S. epidermidis, E. facaelis, and S. pneumoniae ( M I C > 64 pg/ml), the linear amidated peptides showed dramatically improved antimicrobial activities. For example, B a c 2 A - C N had an M I C o f 4 pg/ml against S. aureus, 1 pg/ml against S. epidermidis and 2 pg/ml against E. facaelis.  For b a c 2 A - C N and bac2S-CN the activity against L.  monocytogenes was increased by a factor o f 32, against S. pyogenes and S. mitis by a factor o f 8, and against C. xerosis by a factor of 4. B a c 2 A - C N and bac2S-CN had the same antimicrobial pattern, with only a 2 fold difference in their activities against E. facaelis and S. mitis, the difference that is considered not to be a significant difference for M I C assays (i.e. within the variability o f the assay). These data indicated that introduction o f two O H groups made no difference. On the other hand, b a c S - C N with a single Cys 3 to Ser 3 alteration showed slightly poorer activities (2-4 fold), with exception o f S. pneumoniae (only 2-fold lower MICs) compared to bac2S-CN.  The differences in antimicrobial activities between bac2S and  reduced bacetenecin were not as consistent.  Compared to bac2S, reduced bactenecin  (containing two S H groups) was less active against S. aureus, but was more active against S. pneumoniae, a trend also seen with b a c S - C N (with one S H group).  In general,  amidation of bac2S improved the antimicrobial activities against both Gram positive and Gram-negative bacteria by a factor o f 2-8. The antimicrobial activities o f the reduced forms o f other bactenecin derivatives were also determined (Table IX). Again, the tendency to be Gram-positive selective was  87  observed, yet not as obviously. Most of the linear peptides had decreased or equivalent activities compared with reduced bactenecin, and only 2-4 fold improvements in activities compared to native bactenecin. Bac2R, which has extra positively charged (arginine)  Table VIII: M I C s o f Linear, Amidated Bactenecin Derivatives  M I C (pg/ml) Bacteria Species  Bac2A-CN  BacS-CN  Bac2S-CN  Bac '  E. coli  4  4  2  8  32  >64  P. aeruginosa  8  16  16  4  >64  >64  32  >64  32  8-16  >64  >64  S. aureus  4  16  4  64  16  >64  S. epidermidis  1  2  1  >64  8  8  E. facaelis  2  16  4  >64  8  8  L. monocytogenes  0.25  0.5  0.25  8  1  1  C. xerosis  0.25  0.5  0.25  1  1  0.5  B. subtilis  32  >64  16  >64  64  >64  2  8  2  >64  >64  4  0.25  0.5  0.125  16  16  16  16  8  16  2  0.5  1  2  1  3  Bac 2S  1  Bac-R  1 3  Gram-Negative  S.typhimurium Gram-Positive  S. pyogenes S. mitis S. pneumoniae  * 1. M I C s were also shown in Table V I . Peptide sequences (also see Table III): Bactenecin  RLCRIVVIRVCR  Bac2A-CN  RLARIVVIRVAR-CONH  BaclS-CN  RLSRIVVIRVCR-CONH  Bac2S-CN  RLSRIVVIRVSR-CONH,  Bac2S  RLSRIVVIRVSR  2  2  2. Species and strains are described in Tablell. 3. Bac= Bactenecin Bac-R=Reduced Bactenecin  88  ON OO  I* o  NO  03  A  \P4  h*  CN  NO  A  NO  ^1-  NO  NO  A  A  NO  NO  No A A  CO  oo oo  ©  hf  ho  NO  A  CN NO CN CN NO A cn  NO  A  A  A  O  NO NO NO  A  ICQ  NO  A  A  CN CN CN cn cn  NO  NO  CN cn  A  > CN  CJ  ho  CN  IPQ  hi"  NO  NO CN  CN NO CN CN NO A cn A  cn  NO  A  m CN PH O o3  O oj  O 03  CJ o3  CQ CQ CQ CQ  OO  rt IW  CN  ho  O 03  CQ  o fe  cn o  •g  |pq  O  CD  rt  CD  cn  rt  cn o  • i-H  c  pq 00  o X CU  3  A  00  un  CN cn  o  NO  vo  NO  A  NO  A  A  NO  oo  A  NO  A  NO  A  NO  NO  A  A  NO  NO  A  A  NO  NO  h f NO cn h f CN un o NO o NO A  NO  A  NO  A  un  NO  A  CN o  A  NO  A  00  fl  Cw O  NO  CN  |PQ_  "o3  CD  A  o3  o  CD  NO  03  > "C  fl  •3-  cn o  pq  Q  o  A  o3  CD >  CD  un un  NO  CN CN cn cn  rt  CD  •fl  CN cn  t-H  loo rfl 00  CD  •»—i O  cj  s  ,00^ i l CD  R  C3 oo O R  So!  o  cj  I ca  cu  a  CJ  »R  I* §  ll  £1 cj C3 cj  cj  s oo  O R O  cu R  oo  s cu  R O  £ s  cu  s  §,1  «0  Oo"  oo" oo"  CQ PQ CQ'  residues at both the N - and C-terminus, seemed to have better (2-4 fold) activity than all the rest.  A l l derivatives were relatively inactive against the wild type Gram-negative  bacteria, P. aeruginosa and S. typhimurium, as observed for reduced bactenecin. For E. coli, the derivatives with more positive charges (bac3K,P, bac3K,P,(V), and bac2R) were found to have equivalent antimicrobial activities to native cyclic bactenecin, even though they were linear. BacP, bacW, and bac2R,W were inactive against all three Gram negative bacteria. It seemed that an increase in positive charges, either by amidation or due to the presence of arginine residues, improved the antimicrobial activities for the linear bactenecin derivatives. The effect of positive charges was thus examined further.  C. Positive Charges Eight derivatives of bactenecin were designed to investigate the importance of size, type and topology of positive charges in antimicrobial activity. Six of these peptides had more positive charges than did native bactenecin. The amino acid sequences and numbers of positive charge are listed in Table III and reproduced under each table. Native bactenecin is a type I P-turn loop-structure, with two arginine residues at positions 4 and 9 adjacent to the disulphide bond (Fig. 25).  In order to facilitate the  formation of the disulphide bond, the arginine at position-4 was substituted with a turnpromoting proline residue. In order to maintain the same number of positive charges, leucine at position 2 was substituted by a arginine residue to form bacR,P. BacR,P had positive charges concentrated at the N - and C-termini and a hydrophobic loop (Fig. 26),  90  Figure 25: Computer generated model of bactenecin. This secondary structure model was developed using the program Insight II (Biosystems, San Diego, California) on a Silicon Graphics Indy workstation. The secondary structure was modeled based on energy minimization for 4000 iterations. Three steps of minimization were performed to reach the minimal total energy state: 1) Steepest descent; 2) conjugate gradient; 3) VA09A. Argiriine residues are in red; the cysteine residues are in yellow. Isoleucine and leucine residues are in blue, and valine residues in green; O H group at the C-tenriinus is in white.  91  Figure 26: Computer generated model o f bacR,P. This secondary structure model was developed using the program Insight II (Biosystems, San Diego, California) on a Silicon Graphics Indy workstation. The secondary structure was modeled based on energy mmimization for 4000 iterations.  Three steps o f  rninirnization were performed to reach the minimal total energy state: 1) Steepest descent; 2) conjugate gradient; 3) V A 0 9 A . in yellow.  Arginine residues are in red; the cysteine residues are  Isoleucine and leucine residues are in blue, and valine residues in green; O H  group at the C-terminus is in white; proline residue is in blue.  92  and when modeled seemed to be a good amphipathic molecule.  Bac3R,P was a  derivative of bacR,P with arginine residues added at both the N - and C termini, giving bac3R,P two extra positive charges. Bac3R,P,(V) was made in error during amino acid synthesis with one valine residue in middle of the loop missing. A l l positive charges of native bactenecin are contributed by arginine residues, and not a single lysine residue is present. Bac3K,P was a derivative of bacR,P with three arginine residues replaced by three lysine residues, designed to test the effect of the different basic amino acid side chain. Bac2R was a derivative of bactenecin with two more arginine residues. Bac2IC N and BacP,2R-CN also had one to two extra positive charges compared to the native bactenecin. Among all bactenecin derivatives, Bac2R seemed to have the best antimicrobial activities against Gram-positive bacteria tested (Table X). Compared to native bactenecin Bac2R had >8 fold improved activities against S. epidermidis, C. xerosis, and L. monocytogenes, and 2-4 fold improved activities against E. facaelis, S. pyogenes and S. mitis. Bac3R,P and bac3R,P(V) were the second best, with only about a two fold increase in M I C over bac2R. The absence of the valine residue in the middle of the loop in bac3R,P(V) seemed to make very little difference. A l l three of the above improved peptides had two extra positive charges outside the loop.  93  CD >  CM  PH  O  u co  cu  H  >64  I CN  CN  o  <D  NO T—H  CN ro  o 00 00 NO  NO CN ro NO  NO NO  CN ro CN  r-H  CN CO •<*•  NO CO  o  00  oo  >64  >64  Bac2R  <0.125 >64  00  NO  CN  NO  8-16  >64 >64 r-H  CN  uo ©  >64  oo  NO  <—i  CN  >64  VO  >64  00  >64  oo  >64  oo  >64 >64  CN ro  i—f  >64  00  oo CN ro  >32 N/A N/A N/A N/A N/A N/A  NO  NO  >32 N/A N/A N/A N/A N/A N/A  NO  >64 >64  CN ro  >64  CN o cd PQ NO  >32  i—(  >64  -CN  CN  NO 00  >64 >64  o  >64  60  O  >32 >32  cd  00  >64  PQ i  CN  32-6^  CD  oo  >64 >64 >64  '3 T *  >32  G CD -^-» O  Bac2I-CN PH  >64  _c  Bac3R, P, (V)  CD  Bac3R,  Q  BacR, P  > •c  BacR, P-•CN  cu >  Bac3K,  CO  Bacteria Species Gram-Negative E. coli P. aeruginosa S. typhimurium Gram-Positive S. aureus S. epidermidis E. facaelis L. monocytogenes C. xerosis B. subtilis S. pyogenes & mitis S. pneumoniae  PQ ON  TI-  z  CD  ~  Z  OH PH  Z  U  u  cd cd cd  rt rtCN rt O O O  I  PO CQ pq pq CN O  PH cd  >  1 ID  PH  o  CO  ^rtrtrt  pq pq pq pq pq  ro co ro CN o o o o cd cd cd cd  CN  The introduction o f the turn-promoting proline and movement o f the arginine from position 4 to position 2 (bacR,P) actually had a negative effect on the antimicrobial activities.  The amidation o f this peptide led to restoration o f its activities, which  confirmed that having more positive charges at the N - and C - termini improved the antimicrobial activities. Substitution o f the arginine residues with lysine residues did not seem to have any effect, since bac3K,P had the same M I C values as bacR,P against all tested Gram-positive bacteria except for S. mitis against which bac3K,P had a M I C o f 2 pg/ml, 8 times lower than bacR,P. B a c 2 I - C N and b a c P , 2 R - C N were only tested against three Gram-positive bacteria S. aureus, S. epidermidis, and E. facaelis due to the limited amount o f peptide obtained. They had improved activities only against S. epidermidis. B a c P , 2 R - C N had two arginine residues inserted in the loop, while bac3R,P had them at the ends. At the same time as having improved M I C s against Gram-positive bacteria, bac2R, bac3R,P, and bac3R,P,(V) had the same antimicrobial activities against the Gramnegative bacteria P. aeruginosa, and S. typhimurium as did native bactenecin, with bac2R 2-fold better which was not significant. These three peptides as well as b a c R , P - C N had increased activities by a factor o f 4 against E. coli. B a c R , P - C N , bacR,P, bac2I-CN, and bacP,2R-CN were found to have slightly worse antimicrobial activities against P. aeruginosa and S. typhimurium. BacR,P, bac2I-CN, and bacP,2R-CN had a 2-fold higher antimicrobial activities against E. coli than did native bactenecin. Bac3K,P had the lower activities against all three Gram-negative bacteria, possibly due to the introduction o f both lysine and proline residues.  95  In conclusion, introduction of more positive charges increased the antimicrobial activities, but the position of the positive charges was important. For bactenecin, the positions for addition of charged residues which will improve the antimicrobial activity appeared to be at the N - and C-termini, which form the hydrophilic surface of the bactenecin molecule, while the loop which forms the hydrophobic surface was not an appropriate place for charge addition (Figure 25). The type of positive side chain did not seem to be critical.  D. Hydrophobicity  Two  derivatives  of bactenecin  were made  to determine  hydrophobicity on antimicrobial activities, namely bacP and bacW.  the effect of Hydrophobic  residues such as tryptophan and proline were introduced into the loop since the loop moiety comprised the hydrophobic surface of bactenecin. In their model of bactenecin, Romeo et al. (1988) proposed that bactenecin adopted an antiparallel extended structure forming a bend containing three a-carbon atoms (y turn) at position 7. A proline residue was inserted in the middle of the loop, at position 7 (bacP), to promote the turn as well as hydrophobicity. BacW had an added aromatic tryptophan residue instead. BacP remained relatively inactive against the Gram-positive bacteria S. aureus, S. epidermidis, E. facaelis, B. subtilis, S. pyogenes, and S. pneumoniae, and had similar activities to native bactenecin against C. xerosis, L. monocytogenes and S. mitis, while the activities against Gram-negative bacteria were lost (Table XI). On the other hand, bacW not only retained its activities against Gram-negative bacteria, but it also had comparable  96  antimicrobial activities against Gram-positive bacteria to linear b a c 2 A - C N .  It seemed  that hydrophobicity played a critical role for bactenecin peptides to interact with Grampositive bacteria, which do not have outer membranes. Therefore, hydrophobicity might  Table X I : M I C s o f Hydrophobicity Bactenecin Derivatives M I C (pg/ml) Bacteria Species  BacP  BacW  Bac2R, W  Bac  32  8  2  8  P. aeruginosa  >64  4  2  4  S. typhimurium  >64  4  2  8-16  S. aureus  64  4  2  64  S. epidermidis  64  2  1  >64  >64  8  2  >64  L. monocytogenes  8  1  0.25  8  C. xerosis  2  0.5  0.25  1  B. subtilis  >64  32  64  >64  64  2  1  >64  4  1  0.25  16  >64  16  8  2  Gram-Negative E. coli  Gram-Positive  E. facaelis  S. pyogenes S. mitis S. pneumoniae  1. Peptide sequences (also see Table III): Bactenecin  RLCRIVVIRVCR  BacP  RLCRIVPVIRVCR  BacW  RLCRIVWVIRVCR  B a c W , 2R  RRLCRIVWVIRVCRR  be important for the interaction o f peptides with the cytoplasmic membrane o f bacteria. Tryptophan was a better substitution for improving antimicrobial activity than proline. When comparing bac2R and bacW, it was interesting to find that bac2R had decreased antimicrobial activities against Gram-positive bacteria, except C. xerosis and S.  mitis, than did bacW, while it had a 4-fold increase in activity against E. coli. These results may imply that the effect of positive charges is more important for peptides to interact with the outer membrane of Gram negative bacteria, and that the effect of hydrophobicity is more important for the interaction with the cytoplasmic membrane. Bac2R and bacW, however, had the same M I C against P. aeruginosa and S. typhimurium. Based on the above results I designed bac2R,W which combined the optimal features of bac2R and bacW. It seemed to be the bactenecin derivative with the highest antimicrobial activities against both Gram-negative and Gram-positive bacteria, produced in this study. It had a 4-fold increase in antimicrobial activities against all three Gramnegative bacteria tested, and had at least a 4 -fold, and up to 32-fold increase in activities against Gram-positive bacteria.  E. Disulphide bond  Reduced bac2R,W and bacW lost all activities against all three Gram-negative bacteria, just like the reduced bactenecin, which further confirmed the importance of the disulphide bond for the interaction with the outer membrane of Gram-negative bacterial (Table IX). Interestingly, reduction of the disulphide bond of both bacW and bac2R,W also led to the reduction of activities against Gram-positive bacteria to almost the same level as native bactenecin. This then indicated that there were also some structural requirements for the linear peptides to interact with bacteria and that these were clearly different from the structural requirements of the cyclic peptides.  98  F. Agglutination Activities of Bactenecin and its Derivatives Bactenecin and its derivatives did not lyse human red blood cells, but some of them did cause agglutination of these cells (Table XII). Bac2R,W which had the best antimicrobial activities, also was the most active in agglutinating erythrocytes at 32 pg/ml. This value was at least 4-fold higher than the highest of its MICs. BacW and bac2I-CN also exhibited the same agglutination activity. In general, the reduced forms of the cyclic bactenecin derivatives showed 2-8 fold higher agglutination activities than their oxidized equivalents. Reduced bactenecin also caused agglutination of red blood cells at a concentration of 16 pg/ml, 4 times lower than the hemagglutination concentration of native bactenecin (64 pg/ml). Reduced bac2R,W and reduced bacW also caused agglutination at a lower concentrations than did their disulphide-bridged equivalents, at 8 pg/ml (4-fold) and 4 pg/ml (8 fold) respectively. It seemed that the formation of the disulphide bond inhibited the agglutination of human red blood cells by bactenecin peptides. On the other hand, the linear derivatives bac2A-CN, bacS-CN, bac2S-CN, and bac2S did not agglutinate R B C (>64 pg/ml). BacS-CN, which was only two amino acids different from reduced bactenecin, was non-agglutinating. This may argue that the presence of the SH groups in the cysteine residues of reduced bactenecin may be important in the hemagglutinating effect of reduced bactenecin derivatives. The low agglutinating activities of many bactenecin derivatives, especially those that had a broad spectrum of antimicrobial activities, make these peptides interesting and valuable candidates for drug development.  99  Table X I I : Agglutination Activities of Bactenecin and its Derivatives on Human Red Blood Cells Peptide Bac2A-CN BacS-CN Bac2S-CN Bac2S BacR, P-CN BacR, P Bac3K, P Bac3R, P Bac3R, P, (V) Bac2I-CN Bac2R, P-CN BacP BacW Bac2R Bac2R, W Bactenecin  pg/ml Oxidized Form Reduced or Linear Form >64 32 >64 >64 >64 N/A >64 N/A >64 4 64 4 >64 32 32 N/A >32 N/A 64 32 32 4 64 4 32 8 64 4  G. Summary Bactenecin derivatives were designed based on the general information obtained in previous studies with other classes of peptides. Their antimicrobial and agglutinating activities were determined. The structural elements that were critical for antimicrobial function were thus examined. Their effects on antimicrobial activity of bactenecin is summarized in Table XIII.  More positive charges at the N- and C-termini of the  molecules facilitated the interaction between peptides and target cells.  Increased  100  hydrophobicity in the loop not only increased the antimicrobial activities, but also broadened the antimicrobial spectrum.  The disulphide bond was important for the  interaction with the outer membrane of Gram-negative bacteria. Two interesting candidates of bactenecin derivatives with optimized activities were identified, bac2A-CN and bac2R,W. These two peptides had high activities against both Gram-positive and Gram-negative bacteria, and a low hemagglutination effect.  Table XIII: Influence of Bactenecin Structural Modifications on Antimicrobial Activity  Antimicrobial Activity Gram-negative bacteria Gram-positive bacteria + ++ ++++ + + + +++ ++ ++++  Reduced and Linear Linear/Amidated Positive Charge Hydrophobicity Hydrophobicity/ Positive charge Modifications that resulted in increased activity are indicated by a "+" sign and the numbers of "+" signs represent the relative improvement. Modifications that resulted in decreased activity are indicated by a "-" sign.  101  DISCUSSION A. Overview Polycationic peptides as "endogenous antibiotics" are potential sources of novel antibiotics due to their unique properties.  They are usually amphipathic molecules and  tend to carry multiple positive charges in nature, have antimicrobial activity against a wide range of microorganism including bacteria, fungi and enveloped viruses. They were proposed to have a novel action on bacterial cells as membrane-perturbing agents (Lehrer, 1989; Westerhoff et al., 1989; Skerlavaj et al., 1990), which is different from the conventional antibiotics which act by inhibition of either cell wall or macromolecular synthesis (Neu, 1992). Bactenecin is a cationic loop molecule with only 12 amino acids.  Being the  smallest and simplest disulphide-bonded antimicrobial cationic peptide, bactenecin made an interesting model peptide for this study. Little information is available on its mode of action, and whether it shares a common mechanism with other well-known cationic peptides as a membrane-perturbing agent, or i f it has a unique mechanism due to its small size (too short to span the membrane) and unique loop structure.  Its small size also  makes it a desirable potential therapeutic agent, making easy to produce and manipulate. The disulphide bond could potentially render it protease resistant.  Thus the general aim  of this study was to investigate the mode of action of bactenecin and structure:function relationships in related peptides.  Such a study would provide a better understanding of  the nature of the antibacterial properties of bactenecin, and provide the basis for design of a novel peptide with improved activity and a high therapeutic index.  102  With these aims in mind, two main approaches were taken in this study. First, biochemical assays were conducted to assess the antimicrobial mechanism of bactenecin, especially its interaction with the bacterial membranes including the outer membrane of Gram-negative bacteria and the cytoplasmic membrane. Second, analogs were designed with simple amino acid modifications (substitution and/or addition) to examine the effect of key structural elements (including the disulphide bond, positive charge, ring size, hydrophobicity and amphipathicity) on the antimicrobial activity of bactenecin.  The  toxicity of these analogs was also examined. Besides the main observations, the work in this thesis have raised several issues worthy of discussion and future studies.  B . Recombinant Expression of Cationic Peptides The main focus of this study was the antimicrobial mechanism and the structure:function relationship studies, but first I wished to evaluate the possibility that such peptides could be produced recombinantly, making them accessible for commercial exploitation.  To produce peptide, there are three ways, from the natural source, by  chemical synthesis, and by recombinant expression. Recombinant expression is cheap on a large scale and avoids problems with troublesome amino acids that are difficult to make (e.g. tryptophan and arginine). Recombinant expression also has the potential to be developed as a tool for screening for analogs, to facilitate structure:function studies, and as a tool for incorporating heavy atoms to facilitate 3-dimensional structure studies. The production of bactenecin, as well as two other peptides (indolicidin and apidaecin), was thus attempted by recombinant expression technique.  103  Three  small cationic  peptides,  apidaecin, indolicidin and bactenecin,  were  recombinantly expressed in S. aureus, and bactenecin was also expressed in an E. coli system. Piers et al (1993) were able to successfully express the 26-28 amino acids alphahelical peptides C E M E and C E M A in S. aureus. Although the 33 amino acid peptide H N P - 1 , a (3-sheet structure stabilized by three disulphide-bonds (Piers et al., 1993) was expressed in the linear format, no success in refolding the peptide into its native form was achieved (Hancock, personal communication).  Conversely C E M E and C E M A were  shown to be chemically and biologically identical to their synthetic counterparts, while HNP-1  produced recombinantly did not show any antibacterial activity.  It seemed that  peptides with multiple disulphide bonds could not fold correctly in bacteria. It was the objective o f the first Chapter o f this thesis to extend this technology to smaller peptides (e.g. the 12-amino acid bactenecin, and 13 amino acid indolicidin),. to peptides with highly restricted amino acid compositions (e.g. tryptophan, proline-rich indolicidin and proline, arginine-rich apidaecin), and to loop peptides with a single disulphide bridge (bactenecin).  A s shown here, I was successful in expressing all 3 peptides tried. There  appeared to be no reduction in the amount o f the protein Axationic peptide fusion proteins produced, compared to the amount o f protein A carrier present in control cells. Two products of bactenecin were obtained recombinantly, and identified as an inter-chain disulphide-bonded multimer and an intra-chain disulphide-bonded monomer.  It was not  clear whether the disulphide bonds were formed during the recombinant synthesis in bacteria or during the subsequent purification.  Only one major fusion protein product  was detected, and this seemed to be a monomer (Fig. 3). Since the bactenecin was rather  104  small (less than 1.5 K dalton) compared to its fusion partner (32 K dalton), it might have been difficult for the disulphide bond to be formed at the C-terminus, thus explaining why no dimers or multimers o f fusion protein were detected on S D S - P A G E .  Therefore I  would reason that the oxidation happened during the subsequent C N B r cleavage and purification  procedure,  and both  products  bactenecin) were thus obtained (Fig. 4). product was obtained.  (multimer  and cyclized  monomer o f  A s for indolicidin and apidaecin, only one  Therefore, the S. aureus system,  which permits  efficient  downstream purification due to the secretion o f fusion protein, is acceptable for the production o f different types o f cationic peptides. A n E. coli system was also adopted in this study to optimize the expression and production o f peptides, although an insufficient amount o f pure bactenecin was obtained to do M I C tests. Later, my colleagues Drs. Fidai and Falla did obtain pure bactenecin and indolicidin using this system and showed there was no difference in antimicrobial activity compared to chemically made ones (Zhang et al., 1998).  The E. coli system has been  improved to allow for much higher expression since that time, by utilizing a strong phage T7 promoter and an optimized ribosome binding sequence (Zhang et al., 1998). Apidaecin produced recombinantly showed no activity against Gram-positive bacteria, which agreed with previous studies showing that apidaecin had only activity against Gram-negative bacteria (Casteels et al., 1990). Chemically made indolicidin was shown to be active against E. coli UB1005 ( M I C = 1 6 pg/ml), its mutant D C 2 (4 pg/ml) and P. aeruginosa (64 pg/ml), and it had little activity against Gram-positive bacteria (Falla et al., 1996). These results agreed with the data obtained with recombinantly-made  105  indolicidin (Table V ) . Recombinantly-made, disulphide-bonded bactenecin had 4-8 fold higher M I C s against wild type and mutants o f E. coli and P. aeruginosa compared to chemically-synthesized bactenecin.  This difference may be due to impurities in the  sample, a relatively low efficiency o f disulphide bond formation or inaccuracies in determining the exact concentration o f bactenecin (this was solved later in the thesis by amino acid analysis to accurately determine peptide composition).  Further purification  could be carried out by reverse phase Pep H R 5 column. Recombinantly-made bactenecin did not exhibit any activity against S. aureus ( M I C > 64 pg/ml) and this observation was subsequently confirmed by the chemically-made bactenecin which also had no activity against S. aureus. This result contradicted previous studies (Romeo, 1988) which showed that bactenecin was active with a M I C 1-8 pg/ml against S. aureus.  The different  medium used in the previous studies for M I C determination may partially account for the difference.  L B broth with no salt was used in this study while a Iso-Sensitest broth  (Oxoid, U K ) was used for previous studies.  Another possibility would be that a mixture  of cyclic bactenecin contaminated with linear amidated bactenecin or amidated cyclic bactenecin was used in the previous studies, since the linear amidated bactenecin (Bac2AN H 2 and Bac2S-NH2) was active against S. aureus at a M I C o f 4 pg/ml. Unfortunately, attempts to make pure amidated linear bactenecin (Lin Bac-NH2) and cyclic amidated bactenecin (Bac-NH2), by chemical synthesis, to test this hypothesis, failed. In conclusion, even peptides with restricted or short amino acid sequences, or with single disulphide bonds could be recombinantly produced and had equivalent activity against bacteria to chemically synthesized cationic peptides.  106  C. Antimicrobial Mechanism of Bactenecin 1. The Role of Secondary Structure Bactenecin belongs to a group of cationic peptides with only one disulphide bond. Although the four basic arginine residues permit classification of bactenecin as a cationic peptide, it has a unique, characteristic structure, making it distinct from any other known peptides. It is the smallest cationic peptide with only twelve amino acids, and is too small to span bacterial cytoplasmic membrane to form channels, like the amphipathic a-helical peptide or longer and more complex P-structured defensins, unless a multimer is involved. The disulphide bond makes bactenecin a loop molecule with a hydrophobic ring and positive charges, located at the C- and N-termini, which are brought together by the disulphide bond. Therefore it is interesting to investigate the role of these structural features in the antimicrobial mechanism. The disulphide bond was proven to be a key secondary structure element.  In this study, it was shown that bactenecin was active  against the wild type Gram negative bacteria E. coli, P. aeruginosa and S. typhimurium, whereas the linear unamidated derivative and reduced form were relatively inactive. A l l three forms were equally active against outer membrane barrier defective mutants. This observation indicated that the disulphide bond was important for interaction with the outer membrane as confirmed here. Bactenecin had a better binding ability for LPS than its two linear variants (bac2S and reduced bactenecin) and also permeabilized the outer membrane better, explaining its better activity vs. wild type Gram negative bacteria. Computer modeling of bactenecin with Insightll software (Biosym Technologies Inc.,  107  San Diego, C A ) indicated that bactenecin was a disulphide-bonded loop molecule comprising a long p-turn of hydrophobic residues and a positively charged face constructed from the C- and N-terminal portions of the molecule (Fig. 25). The model was consistent with the C D spectral studies (Fig. 10), which indicated that bactenecin existed as a rigid P-turn loop molecule regardless of its environment.  Such a  conformation, may make bactenecin a more amphipathic molecule than the unstructured linear and reduced form which exist in solution as random structures. This could explain why bactenecin interacted better with the negatively charged LPS than its linear and reduced forms. Alternatively since the loop brings together the 2 N-terminal and 2 Cterminal arginines, it could be that the higher local charge density is important for disrupting the divalent cation bridging of LPS at the outer membrane surface. Compared to polymyxin B and two a-helical peptides C E M E and C E M A (Piers, 1994), bactenecin was a relatively weak outer membrane permeabilizer. Bactenecin was too small to span the membrane and form pores or channels unless a multimer is involved. Therefore it was of interest to know how this molecule interacted with the cytoplasmic membrane, which was believed to be the final target of many cationic peptides (Lehrer, 1989). If bactenecin interacted with the cytoplasmic membrane, it should at a minimum make it proton-leaky, and thus dissipate the membrane potential. The cytoplasmic membrane depolarization assay results showed that bactenecin had only minor effects on membrane potential at M I C concentrations, whereas the maximal effect was only seen at 4 fold the MIC.  108  Linear variants of bactenecin adopted (3-structure in the presence o f liposome. Despite their equivalent M I C value against E. coli D C 2 , the pattern o f interaction of bactenecin and its linear variants with the cytoplasmic membrane was quite different. The linear variants dissipated the cytoplasmic membrane potential at concentrations as low as 0.125 pg/ml, and rapid kinetics were observed.  This pattern was similar to that  seen with the a-helical peptide C E M E , which suggest that they act similarly. Planar bilayer studies showed that the linear variants and the native bactenecin had different channel forming activities, which agreed with the cytoplasmic membrane permeabilization results.  The loop bactenecin was poorly active in model membranes,  requiring high voltages to initiate conductance events and forming smaller channels compared to its linear variants (reduced bactenecin and Bac2S) (Wu and Hancock, submitted). The  different  conformations  that these bactenecin  peptides  adopted  when  interacting with hydrophobic membranes may be responsible for these differences. Linear variants with a more flexible structure were able to interact with the inner membrane in a manner that was  fundamentally different from that o f the native  bactenecin with a rigid cyclic structure. The lethal step for the native bactenecin may be quite different from the model proposed for classical peptides like defensin and cecropin, while linear variants may function by a similar channel-forming mechanism.  Linear  variants are also too small to span the membrane unless multimers were involved. The possibility that these peptides  may change  their conformation (disulphide-bonded  bactenecin b e i n g r e d u c e d or linear forms b e i n g o x i d z e d ) once i n s i d e the c e l l c a n not be excluded. In c o n c l u s i o n , secondary structure p l a y s a k e y r o l e i n the interaction o f bactenecin w i t h bacterial c e l l s . T h e results argues for a distinct m e c h a n i s m o f a c t i o n for this peptide, s u c h as the ones p r o p o s e d for p o l y c a t i o n i c a m i n o g l y c o s i d e s ( H a n c o c k , 1981). mechanisms  o f action  have  been  suggested  for  a n t i m i c r o b i a l peptides,  Other  including  s t i m u l a t i o n o f auto l y t i c e n z y m e s ( C h i t n i s et a l , 1990), interference w i t h bacterial D N A and/or p r o t e i n synthesis (Lehrer, 1989; B o m a n et a l , 1993), i n h i b i t i o n o f D N A synthesis l e a d i n g to f i l i a m e n t a t i o n ( S u b b a l a k s h m i et a l . , 1998), or general b i n d i n g to and i n h i b i t i o n o f c e l l u l a r n u c l e i c acids (Park et a l . , 1998).  2. Is the M e m b r a n e the O n l y and F i n a l Target of Cationic Peptides? L e h r e r et a l . (1989) s h o w e d that defensins p e r m e a b i l i z e d b o t h the outer and inner m e m b r a n e o f E. coli, and that the inner m e m b r a n e p e r m e a b i l i z a t i o n and c e l l death were two  closely  linked  events.  Therefore  they  proposed  that  the  inner  p e r m e a b i l i z a t i o n w a s the lethal step o f the k i l l i n g a c t i o n o f c a t i o n i c peptides.  membrane However, a  m i x t u r e o f H N P 1 - 3 ( l O O p g / m l , at a m o l a r ratios o f 1:1:0.5 instead o f M I C amounts) were used i n this study, this c o m p l i c a t i n g the results. L a t e r ex v i v o studies w e r e interpreted as i n d i c a t i n g that the f o r m a t i o n o f m u l t i m e r i c pores w a s responsible for i n n e r  membrane  p e r m e a b i l i z a t i o n ( K a g a n et a l . , 1990; H i l l et a l , 1991; S c h w a r z et a l . , 1992; W i m l e y et al., 1994; M a t s u z a k i et a l . , 1994; R e x , 1996). T h e f o r m a t i o n o f i o n channels w o u l d cause the leakage o f protons or other ions and depolarize the m e m b r a n e potential.  However,  110  studies regarding the inner membrane activity were mainly done on model membrane. T o investigate this event from the microbiological, instead of biophysical point of view, an assay was developed by taking advantage of an outer membrane-defective mutant E. coli D C 2 and a membrane potential indicator DiSC3-(5).  The ability of peptides  representing different secondary structures to depolarize the membrane were examined. If the final and only target of cationic peptides was the inner membrane, one would expect that the killing concentrations of peptides (MICs) would lead to the complete disruption of the inner membrane. It was surprising to find that there was no correlation between the ability to dissipate the membrane potential and the concentration leading to cell death. A t their M I C s , different degrees of membrane potential dissipation were observed. For example, the alpha-helical peptide CP26 (Fig. 16) failed to cause any dissipation of the membrane potential, the beta-structured peptide Gram4112 (Fig. 17) and two extended-structured peptide C P 1 1 C N and C P 1 0 C N (Fig. 18) caused partial depolarization, whereas the loop-structured peptide bactenecin (Fig. 19) caused only partial and very slow depolarization.  The concentrations required to cause maximum  dissipation of membrane potential varied also; some peptide required concentrations much lower than the M I C (e.g., two P-structure peptides Gramicidin S and Gram 474), whereas some required concentrations higher than the M I C (e.g., CP26). Gram474 caused maximum depolarization at 4 pg/ml (8 fold lower than its M I C ) , while CP26 did this at 4 pg/ml (8 fold higher than its M I C ) . These observations suggest that the killing mechanism may not be as simple as previously proposed, and multiple mechanisms may be involved. For peptides like CP26,  111  very few peptide molecules that bind to the inner membrane will form channels or pores. It is proposed that these will flip-flop across the membrane without formal channel formation and in this way get into the cells and cause subsequent events which contribute to the cell death (Wu et al., submitted).  For Gram474, dissipation o f the membrane  potential is clearly not enough to kill the bacterium and other targets must be required. Interestingly, no cell lysis was observed for any o f the peptides studied, and there was no change in optical density over time, when the tests were conducted for either CP26 and Gram474. Inhibition o f synthesis o f macromolecules or precipitation o f D N A and R N A , since D N A and R N A molecules are strongly negatively charged polyanions, would be possible mechanisms that might occur after entry o f cationic peptides into the cytoplasm.  3. Do A l l Cationic Peptides Interact with the Bacterial Cytoplasmic Membrane by the Same Mechanism? The results of the cytoplasmic membrane permeability studies also raised another question. D o all cationic peptide interact with this membrane by the same mechanism? Different patterns o f membrane potential dissipation were observed as discussed above, especially among peptides with different structures. Again CP26 and Gram474 would be two examples that represent the extremes.  The extended-structured peptides ( C P 1 1 C N  and C P 1 0 C N ) as well as bactenecin and its derivatives (bac2S and reduced bactenecin) are only 12-13 amino acids long, and as such they are not long enough to span the membrane. The bilayer membrane of an E. coli cell is approximately 5.4 nm to 6.5 nm thick (Park, 1987). In order for a peptide to span this distance as an a-helix it must be 20  112  amino acids or more in length or as a P-strand 14 amino acids (Eisenberg, 1982). Thus these peptides may interact with the membrane in a way different from the longer peptides (e. g. a-helical peptides).  Longer peptides are potentially capable of forming  regular channels by multimerization, like the model proposed for defensin and cecropin. In contrast,  shorter peptides may just aggregate in the membrane thus disrupting  membrane integrity (Wu et al., submitted).  D. Design of Novel Peptides with Improved Activity 1. Structure:Function Relationships Model peptides were designed to study the structure:function relationships in bactenecin-related peptides.  Three structural elements were examined, namely basicity,  hydrophobicity and loop-structure.  B y modifying these elements, the antimicrobial  activities were either improved or decreased.  Computer modeling of the best and worst  molecules were conducted in an attempt to explain the results at the level of assumed three dimensional structure. Increasing the numbers of positive charges at the C - or N- terminus either by including more arginine residues, or by amidation of the carboxyl terminus, increased antimicrobial activity.  B a c 2 S - C N had better activity against both Gram-positive and  Gram-negative bacteria than bac2S by at least 2 fold, and up to 128 fold (Table VIII). Bac2R with two extra arginines at C - and N-terminus also had better activity compared with the native bactenecin. bactenecin (Fig. 25).  This could be explained by the molecular modeling of  Cyclic bactenecin is an amphipathic molecule composed of a  113  hydrophobic loop and two positive charged ends that are brought in proximity to one another by the disulphide bridge. When the molecule binds to the negatively charged bacterial surface, the two ends will probably come in contact with the negatively charged bacterial surface first. Therefore increasing the positive charges at the terminus would presumably facilitate the interaction of peptides with the negatively charged cell surface. There were two kinds of residues which were introduced into the middle of the loop at position 7 to promote a turn (and make disulphide bond formation easier), proline (BacP), and tryptophan (BacW). BacP had worse activity, while BacW had improved activity. Fig 27 shows that insertion of the proline residue changed the direction of one arginine residue and the amphipathicity of bactenecin was destroyed, explained the decreased activity. Insertion of a tryptophan residue on the other hand maintained the amphipathic properties of bactenecin (Fig. 28 and 29). Thus, although bactenecin can tolerate larger ring structures, it is very important as to which residue is employed. Introduction of a proline residue at position 4 also decreased the antimicrobial activity against P. aeruginosa and S. typhimurium, although the molecule seemed still to be an amphipathic molecule by computer modeling. This may be because the two arginine residues (position 1 and 13) were brought too close to each other (Fig 25).  E. Potential Application of Bactenecin There are several potential advantages of bactenecin and derivatives (Bac2A-NH2 and Bac2R, W) as antibiotics. They are relatively small, only 12-15 amino acids long. They would be easier to manipulate. They demonstrate no toxicity against human red blood cells. Certain peptides e.g. bac2A-CN and bac2R,W have a broad antimicrobial 114  spectrum against both Gram-negative and Gram-positive bacteria, and especially against several clinically important pathogens.  Furthermore, the disulphide bond may render the  peptides protease resistant, because the secondary structure maintained by the disulphide bond may make the molecule less accessible for proteases. MSI-78 is a 22-residue magainin analogue that has been in human Phase Ilb/III clinical trials. Four applications have been suggested for MSI-78 as a potential human therapeutic agent, "as a broad-spectrum topical agent; as a systemic antibiotic, as a wound healing stimulant, and as an agent against cancer" (Jacob and Zasloff, 1994). B a c 2 A - C N and bac2R,W certainly have the potential to be developed as broad spectrum antimicrobials.  Their anti-cancer, and anti-virus activity and other therapeutic function  are also worth investigating in future studies.  F . Future Studies The optimal use of these peptides as antimicrobials will require full and detailed knowledge of their mode of action and the structural elements important in their activity and specificity (Nissen-Meyer and Nes, 1997).  Therefore future studies should still  concentrate on these aspects. A s this study showed, cyclic bactenecin may have cellular targets (e.g. D N A , protein etc.) other than bacterial membrane.  It will be very important to identify these  targets and to understand the mode of action in the future.  Bactenecin derivatives with  both improved activity (bac2A-CN and bac2R,W) and worse activity (bacP and bacP,R) have been identified in this study.  Thus the biochemical assays described in this study  (e.g. the outer and cytoplasmic membrane permeability assays, dose and time-dependent  .  115  kinetics of killing assay) should be applied to both types of derivatives in an attempt to explain these differences. The results obtained will help to further understand the effect of simple structural modifications on the antimicrobial mode of action. Secondary structure studies on these derivatives by circular dichroism in different environment (aqueous, hydrophobic, in the presence of liposome or LPS) and by 2-dimensional N M R should be done in conjunction with the antimicrobial mechanism studies. variants of bactenecin and cyclic bactenecin may have fundamentally  The linear different  mechanisms, as shown in this study. Thus bac2A-CN, a linear peptide and bac2R,W, a cyclic peptide may also have different modes of action and different approaches may be taken to study their modes of action.  The factors that determine the different  antimicrobial spectra of bactenecin and its derivatives against Gram-negative and Grampositive, are poorly understood. This is another aspect that worth looking into. In summary, this study has provided some insight into the mode of action of bactenecin and its structural requirements for activity. There is plenty of work that needs to be done to fully understand the antimicrobial nature of this peptide and to its derivatives and develop a useful antimicrobial agent.  116  Figure 27: Computer generated model of bacP. This secondary structure model was developed using the program Insight II (Biosystems, San Diego, California) on a Silicon Graphics Indy workstation. The secondary structure was modeled based on energy rrunimization for 4000 iterations. Three steps of mirrimization were performed to reach the minimal total energy state: 1) Steepest descent; 2) conjugate gradient; 3) VA09A. Arginine residues are in red; the cysteine residues are in yellow. Isoleucine and leucine residues are in blue, and valine residues in green; O H group at the C-tenninus is in white, proline residue is in blue.  117  Figure 28: Computer generated model of bacW. This secondary structure model was developed using the program Insight II (Biosystems, San Diego, California) on a Silicon Graphics Indy workstation. The secondary structure was modeled based on energy minimization for 4000 iterations. Three steps of minimization were performed to reach the minimal total energy state: 1) Steepest descent; 2) conjugate gradient; 3) VA09A. Arginine residues are in red; the cysteine residues are in yellow. Isoleucine and leucine residues are in blue, and valine residues in green; O H group at the C-terrninus is in white, tryptophan residue is in blue.  118  Figure 29: Computer generated model of bac2R, W. This secondary structure model was developed using the program Insight II (Biosystems, San Diego, California) on a Silicon Graphics Indy workstation. The secondary structure was modeled based on energy nrinimization for 4000 iterations. 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