Strategies for New Antimicrobial Proteins and ... - IngentaConnect

Current Pharmaceutical Design, 2002, 8, 671-693

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Strategies for New Antimicrobial Proteins and Peptides: Lysozyme and Aprotinin as Model Molecules Hisham R. Ibrahim1*, Takayoshi Aoki1 and Antonio Pellegrini2 1Department

of Biochemistry and Biotechnology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan and 2Institute of Veterinary Physiology, University of Zürich, Winterthurerstrasse 260, 8057-Zürich, Switzerland Abstract: The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.

INTRODUCTION Microorganisms have coevolved with humans and animals; most often the relationship is mutually beneficial. In instances where there is competition, e.g. diseases, both the microorganisms and humans have had an opportunity to develop defensive strategies. Bacteria continually develop resistance to antimicrobial agents reaching at present alarming levels. This situation is considered now as one of the major public health problems, thus for the safety of drug and food systems, there is a strong need to continually develop new antimicrobial agents. During the past few decades, the discovery of new antimicrobials has relied on the systematic screening of natural products and synthetic chemicals. Several antimicrobials have been discovered and used either as drugs (antibiotics) or in foods as preservatives. Some major factors to consider when selecting an antimicrobial include: (1) the antimicrobial spectrum and physicochemical properties of

*Address for correspondence to that author at the Department of Biochemistry and Biotechnology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. Tel : +81 (99) 285-8658; Fax: +81 (99) 285-8525; E-mail: [email protected]

1381-6128/02 $35.00+.00

the compound; (2) the safety of the compound in its intended use; and (3) the type of microorganisms present [1]. With all these factors to consider, more than one antimicrobial may be needed. Therefore, any success in engineering the potency and specificity of a safe antimicrobial agent to be active in killing varying types of bacteria would be an important contribution to modern biotechnology. In this review, we will discuss how we might use the knowledge of naturally occurring physiological processes in vivo to potentially engineer the antimicrobial specificity of a safe and widely distributed proteins, such as hen egg-white lysozyme and aprotinin, by which the authors collected experience in the last few years. The effectiveness of lysozyme in food systems and therapy is based on its ability to control the growth of susceptible bacteria. One of the mechanism proposed by which lysozyme kills the sensitive bacteria is by the degradation of the glycosidic β-linkage between the Nacetylglucosamine (NAG) and the N-acetylmuramic acid (NAM) of the peptidoglycan layer in the bacterial cell walls [2]. Such a mechanism of action of lysozyme is, unfortunately, limited to certain Gram-positive bacteria. Invasive Gram-negative bacteria, which cause about one-third of all bacterial infections, are resistant to the lytic action of © 2002 Bentham Science Publishers Ltd.

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lysozyme. The peptidoglycan layer in Gram-negative bacteria is protected from the lytic action of lysozyme because the outermost surface (outer membrane, OM) of the bacteria functions as a permeability barrier [3, 4]. Hence, in order for lysozyme to be effective against Gram-negative bacteria, it must overcome the OM's permeability barrier. One of the possibilities that this barrier could be circumvented is by equipping the lysozyme molecule with a hydrophobic carrier, which could mediate its interaction and insertion into membrane. This would facilitate its delivery into the site of its action (the peptidoglycan) which successively would lead to damage of the cytoplasmic membrane, CM, (the sensitive killing site). Another mechanism of the bactericidal action proposed for lysozyme is independent of its enzymatic activity but attributed mainly to its cationic and hydrophoibic properties. This mechanism of action is supported by the fact that denaturated lysozyme lacking enzymatic activity is still able to inhibit the bacterial growth [5,6] .

Bacteria are classified as Gram-positive or Gram-negative according to their membrane structures which influence the response to the Gram-staining procedure. The basis for this difference relates to the bacterial cell wall. The envelope structure of a typical Gram-positive (left) and a typical Gram-negative (right) cell are different (Fig. 1). Bacteria show a surprising degree of complexity in their envelopes. The layers of the cell envelope lying external to the cytoplasmic membrane (CM) are referred collectively to the cell wall which is present in both Gram-positive and Gramnegative organisms. The thick peptidoglycan layer constitutes most of the Gram-positive cell wall, which is sensitive to the action of lysozyme and penicillin.

Before exploring our novel strategies to engineer the antimicrobial function of lysozyme, it will be helpful to discuss some background issues. First, what is the difference in the membrane structures of Gram-positive versus Gramnegative bacteria? Second, how do different kinds of antimicrobials work? And third, what are the structural requirements for the antimicrobial properties of lysozyme and aprotinin ?

The cell walls of Gram-negative bacteria have a more complicated structure. They possess an additional layer and externally the cell appears convoluted (Fig. 1, right). Two membranes are present in these bacteria strains. Outside of the cytoplasmic membrane (CM) there is an open area called the periplasmic space. Beyond this a thin layer of peptidoglycan finds place. Finally, external to the peptidoglycan is an additional membrane, the outer membrane (OM). The cell wall of Gram-positive bacteria consists mainly of the peptidoglycan layer and lacks the OM. The peptidoglycan is much thicker (20-80 nm) than in the Gram-negative bacteria (2 nm) and externally it has a smoother appearance (Fig. 1, left). In Gram-negative bacteria, the cell wall includes the OM, lipoprotein, lipopolysaccharide (LPS), and peptidoglycan layers (Table 1). Peptidoglycan is a rigid layer that is found in both cell types and is composed of a overlapping lattice of 2 sugars NAG and NAM that are crosslinked by amino acid bridges ( Fig. 2) [18]. In Gram-positive bacteria the peptidoglycan is a heavily cross-linked woven structure that wraps around the cell. It is a very thick layer with peptidoglycan accounting for 50% of the weight of the cell and 90% of the weight of the cell wall (Table 1). In Gram-negative bacteria the peptidoglycan is much thinner with only 15-20% of the cell wall being made up of peptidoglycan and this is only intermittently cross-linked. It is covalently attached to a lipoprotein in the OM. In both cases the peptidoglycan can be thought of as a strong, woven mesh that maintains the cell's shape. It is not a barrier to solutes, the openings in the mesh are large and almost all types of molecules can pass through. The cell wall determines the shape of the cell and also protects cells from osmotic lysis. Without a wall the cell would swell and burst. Any cell that has lost its cell wall, either artificially or naturally, becomes amorphic, i.e., without a defined shape. The cell wall is the site of action of many important antibacterial agents (Table 1). Penicillin inhibits cells wall synthesis (cross-linking). Lysozyme attacks peptidoglycan, by hydrolyzing the NAG-NAM linkage (Fig. 2).

BASIC TYPES ENVELOPES

HOW DO WORK?

We have succeeded in achieving the goal of engineering new antimicrobial substances by four different types of design. In two approaches, we modelled the lysozyme molecule on the structural properties of natural membranefusing proteins such as the amphitropic proteins [7] or the fusogenic proteins [8]. These modifications were carried out either by fatty-acylation of Lys residues [9, 10], or by genetic fusion of hydrophobic peptides of different lengths to the C-terminus of lysozyme [11-13]. The third approach was based on using the basicity of lysozyme and its tendency to interact with the OM's lipopolysaccharides (LPS) to facilitate the localization and concentration of an edible preservative, phenolic aldehyde, perillaldehyde [14], at the killing site (CM) of resistant bacteria [15]. The fourth approach, followed to design bactericidal substances from lysozyme or aprotinin by means of proteolytic enzymes and investigate the bactericidal properties of their peptide fragments [16,17]. Once a bactericidal fragment is characterized, modifications of its amino acid sequence is performed to generate a stronger bactericidal derivatives of the peptides. Following the latter approach we were able to develop a rational design of short bactericidal peptides derived from lysozyme and aprotinin.

OF

BACTERIA

AND

THEIR

Knowledge of the structure of a bacteria as well as the properties of an antimicrobial compound helps in understanding how a microbe resists destruction and allows researchers to develop the proper strategy for designing potentially novel bactericidal agents.

ANTIMICROBIALS

GENERALLY

The common antimicrobial agents kills the bacteria through four major mechanisms of action. There are numerous antibacterial agents that target the cell wall because mammals do not synthesize cell walls and therefore

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Fig. (1). Comparison of the cell membranes structures of Gram-positive (left) and Gram-negative (right). View of the whole cells (A) and cross section in their envelopes (B). In Gram-negative bacteria the aqueous space (periplasm) between the cytoplasmic and outer membrane is shown.

are immune to the toxic effects of these agents, e.g., penicillin and lysozyme. The cell wall acts as a corseting structure protecting the cell against osmotic lysis. Agents that destroy the wall (lysozyme) or prevent its normal synthesis (penicillin) normally bring about lysis leading to death. Other antimicrobials act by damaging DNA or preventing its synthesis, e.g., alkylating agents and radiation, while others inhibit protein synthesis depriving cells of proteins and enzymes necessary for biological reactions, e.g., aminoglycosides such as streptomycin and tetracyclines. The fourth group of antimicrobials act like detergents on the cell membrane, e.g., polymyxins, cecropin and colicin disrupt the integrity of the lipid bilayer membrane. However, the ability of bacteria to develop resistance to antimicrobial agents is an important factor in their control.

There are many different mechanisms by which bacteria might exhibit resistance to antimicrobials. These includes: (1) production of enzymes that inactivate the antimicrobial; (2) changing their permeability to the antimicrobials; (3) developing an altered structural target for the antimicrobial; and (4) developing an altered metabolic pathway that bypasses the reaction inhibited by the antimicrobial. All of these mechanisms of bacterial resistance occur in nature. However, bacteria are unlikely to develop resistance to antimicrobials such as α-toxin and colicins that act on the cell membrane by irreversibly inserting themselves into the lipid bilayer of the CM, thus forming a pore into it (poreforming peptides, PFP). To design a strong antimicrobial molecule that is active in killing both Gram-positive and Gram-negative bacteria, it must be targeted at the CM. For Gram-negative bacteria, it

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Table 1.

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Difference Between Gram-positive and Gramnegative Bacteria. A Comparison Related to Their Cell Wall Types Property

Gram-positive

Gram-negative

Number of Layers

1

2

Wall Thickness

20 ~ 80 nm

~10 nm

Peptidoglycan Content

> 50 %

10 ~ 20%

Lipid / Lipoprotein Content

0~3%

58 %

Protein Content

0%

9%

Sensitive to Penicillin

(+)

(–)

Sensitive to Lysozyme

(+)

(+)

must be able to penetrate the OM, hydrolyzing the peptidoglycan layer and then insert itself into the lipid bilayer of the CM. Lysozyme is probably the best candidate to fulfill these requirements. Operationally it mimics the action of the membrane active fusogenic bacterial toxins, and unlike these toxins, it can be used in food or drug systems [8]. The inherent bacteriolytic action of lysozyme is discussed below [2, 18, 19]. LYSOZYME AND ITS MODE OF ANTIMICROBIAL ACTION Lysozyme ( also known as muramidase) was discovered in the nose secretion by Fleming in 1922 [20]. It is an

enzyme able to hydrolyse the peptidoglycan layer of cell wall of some Gram-positive bacteria causing in this way the bacterial death. This property and the fact that it is present in the neutrophil granulocytes, monocytes and macrophages as well as in many biological fluids and tissues of a large number of living organisms makes lysozyme an important component of the non-specific defence mechanisms of the organisms and one of the humoral factors of the innate immunity in animals [21-27]. Lysozyme is a basic protein of 14.4 KDa ( 129 amino acid residues), a molecular weight, which is low compared with most of proteins and enzymes. This character and the fact that it can be produced in large amount with low prime costs from the chicken egg white has made it an ideal enzyme molecule to investigate the catalytic properties of the enzymes. Indeed chicken egg white lysozyme was the first protein which was sequenced and the first enzyme whose three-dimensional structure was analysed by X-ray crystallography [2]. The structure of lysozyme consists of two domains in which one is composed predominantly of helices structures whereas the other domain contains the β−sheets. The two domains are separated by a deep cleft which stretched across the whole molecule. The natural lysozyme substrate is the petidoglycan, a gigantic insoluble molecule, present in the prokaryotes cell only, which surrounds the cell as a network. Its structure contains NAG, NAM and a tetrapeptide which contains some Damino acids ( Fig. 2) . Four disulfide bridges between the residues 64-80, 76-96, 6-127 and 30-115 cross-linked the molecular structure of lysozyme. The enzymatic activity of lysozyme is lost when more than two disulfide bridges are destroyed. Two forms of lysozyme which differ from each other in molecular weight and amino acid sequence have been found in avian egg white. One form called c-lysozyme

Fig. (2). Primary structure of the peptidoglycan polymer present in the cell wall of bacteria. The peptidoglycan polymer consists of a large number of subunits N-acetylglucosamine (G) and N-acetylmuramic acid (M) with attached a peptide chain . The arrows represent the point of attack of lysozyme whereas the dashed line represent the part of the peptidoglycan structure sensitive to penicillin


is typified by lysozyme found in chicken egg white, whereas the other form, g-lysozme is represented by the lysozyme found in the Embden goose [2]. It has been believed till recently that lysozyme as a bactericidal agent is effective against only Gram-positive bacteria because as muramidase it is able to lyse these bacteria strains acting on the peptidoglycan polymer of the cell wall. Gram-negative bacteria can not be lysed directly by lysozyme, presumably because of the protective function of the outer membrane shielding the peptidoglycan layer from the environment. Gram-negative bacteria are assumed to be lysed by lysozyme only after that the permeability of the outer membrane has been altered so that lysozyme gains access to the peptidoglycan layer to act there as muramidase [28-31]. During our investigations on lysozyme we could


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show that Gram-negative bacteria are killed by lysozyme but not lysed [32,33]. This results have been more recently confirmed from the investigations of lysozyme on E. coli using either cryoelectromicroscopy technology or the modified E. coli ML-35p strain [34]. Ultrastructural investigation indicate that lysozyme does not affect the bacterial morphology but impairs stability of Gram-negative bacteria [33,34]. Susceptible bacteria to the enzymatic action of lysozyme are not lysed when they are in an osmotically balanced medium [35]. As shown in Fig. 3, in order for lysozyme to kill the susceptible Gram-positive bacteria, the rate of peptidoglycan degradation must be several folds higher than the opposite rate of peptidoglycan synthesis by bacteria. Also it is necessary to maintain an osmotically unbalanced medium to assure the bursting of the cell upon hydrolysis of the cell wall, otherwise death does not occur.

Fig. (3.) Lysozyme attack of the cell walls of Gram-positive bacteria. Cell death occurs by the lytic action of lysozyme on peptidoglycan only when in low-osmotic-strength media, or when the rate of the synthesis and polymerization processes for new peptidoglycan formation is slower than the lysozyme catalyzed degradation.

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On the other hand, lysozyme is known to be much less effective against Gram-negative bacteria including food-borne pathogens, thus limiting its potential incorporation into food and drug systems. If lysozyme is converted into a membrane-penetrating form its bacteriolytic action might include Gram-negative bacteria. Dual antimicrobial action for lysozyme could be achieved if the enzyme could be conjugated to a lipidsoluble membrane active antibiotic that would deliver it to the CM.

STRATEGIES FOR DESIGNING NEGATIVE LYSOZYME

ANTI

The other option was based on the fact that many of the hydrophobic phenolic aldehydes of edible green leaves exhibit weak antimicrobial action when targeted to the CM of bacteria, the aldehydes are immobilized in the hydrophobic OM of Gram-negative bacteria or are excluded by the hydrophilic cell wall of Gram-positive ones. Therefore, we postulated that a potent bactericidal molecule can be engineered by conjugating such a hydrophobic antibiotic to the catalytic function of the highly hydrophilic lysozyme, considering that lysozyme disrupts the LPS layer of the OM of Gram-negative bacteria. Thus, lysozyme may facilitate the delivery and concentration of the weak hydrophobic antibiotic to the CM (Fig. 4B).

GRAM-

Our strategies to convert lysozyme to be effective against Gram-negative bacteria, without detrimental effect on its inherent action against Gram-positive ones, were based on two options, which are elucidated in Fig. 4. In the first option, lysozyme was converted into a membranepenetrating form, by adding a surface-exposed hydrophobic domain, to mediate its fusion into the OM and deliver it to the site of action (the peptidoglycan) and subsequent insertion into the CM (Fig. 4A). This goal was achieved by using two approaches: (1) chemical modification of lysyl residues with different lengths of saturated fatty acids, an amphitropic protein-mimetic approach (amphitropic proteins are lipid-binding proteins found in vivo which can traverse cell membranes reversibly [7]); and (2) genetic fusion of hydrophobic peptides of various sizes to the C-terminus of lysozyme, a fusogenic protein-mimetic approach (fusogenic proteins are natural proteins which can pass through biological membranes [8].

FATTY-ACYLATION STRATEGY FOR DESIGNING ANTIMICROBIALS Given the above background, hen egg-white lysozyme (NLz) was modified to various degrees with stearic (C18), palmitic (C16), and myristic (C14) acids, which are all saturated fatty acids, are the predominant fatty acids found attached to proteins in living cells that mediate a number of functions, including membrane interaction, translocation, and promoting protein-protein interaction [37]. The fatty acids were covalently attached to lysyl residue of lysozyme by base-catalyzed ester exchange [38]. The degree of incorporation was controlled by adjusting the molar ratio of the N-hydroxysuccinimide (NHS) ester of the fatty acid to lysozyme, based on six available Lys residues per molecule [9, 10]. The preparations were performed at a mole ratio of the respective fatty acid to lysozyme of 1, 2, 3 or 4 to obtain stearoylated (S-Lz), palmitoylated (P-Lz) and myristoylated (M-Lz) lysozyme with various degrees of

Fig. (4). Strategies for developing antimicrobially active lysozyme for Gram-negative bacteria. (A) Equipping lysozyme with a hydrophobic carrier, by acylation or genetic fusion, to enable the molecule to permeate (arrows) the cytoplasmatic membrane (IM). (B) Delivering a membrane-active phenolic compound by attaching it to lysozyme. The white region on lysozyme represents the active site. IM, OM, and PG refer to the inner- and outer-membrane, and peptidoglycan, respectively.



modification, as illustrated in Fig. 5. The stoichiometry of the modified lysozyme was confirmed by measuring the TNBS-non available ε-amino groups of the lysozyme. The degree of modification with palmitic acid displayed an approximately linear correlation with increasing mole ratio of palmitic acid to lysozyme (Table 2). Incorporation of myristic acid exhibited a sigmoidal curve, and the amount of incorporation was considerably less than that of palmitic acid. On the other hand, incorporation of stearic acid into lysozyme showed a progressive increase. The results suggests that the amount of incorporation was in the order of palmitic> stearic> myristic acid. However, in all cases at least two residues of fatty acid were incorporated, the optimum degree of modification found in vivo [37]. There was a gradual decrease in the enzymatic activity of S-Lz and P-Lz lysozymes as the degree of incorporation increased (Table 2). It is noteworthy that modification with approximately one to two fatty acids per molecule of lysozyme maintained a reasonable enzymatic activity (more than 60%).

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microorganism (E. coli K-12). Bacteria were incubated with the fatty acylated lysozymes for 10 min at 25 °C and the CFU were determined on nutrient agar plates. A significant decrease in the viable cell numbers was observed with stearoylated and palmitoylated lysozymes at one residue of fatty acid. Monomyristoylated lysozyme exhibited less antibacterial activity. Further modification toward two residues of fatty acid per molecule of lysozyme increased the antibacterial activity of palmitoylated lysozyme but decreased that of stearoylated lysozyme. Dimyristoylated lysozyme showed a marginal increase in antibacterial action. Higher degrees of modification led to loss of antibacterial activity for all three fatty acids. Interestingly, a linear correlation was observed between the decrease in the survival of E. coli and the length of the carbon chain (from 14 to 18 carbon atom) for modified lysozyme with a single residue of fatty acid. The lysozyme derivatives incorporating one stearate or two palmitate residues were the most potent lysozyme types against E. coli K-12. Localization of Modified Lysine Residues

ANTIMICROBIAL ACTIVITY ACYLATED LYSOZYME

OF

FATTY-

The capability of these lysozyme derivatives to kill Gram-negative bacteria was tested against a representative

Localization of the modified lysine residues by peptide mapping [10] of the di-palmitoylated lysozyme, the most potent antimicrobial derivative, indicated that Lys-33 and Lys-97 are the susceptible nucleophiles. The two

Fig. (5). Schematic representation of the preparation of covalent modification of lysozyme with fatty acids of different lengths. NHS-, N-hydroxysuccinimide ester. C14, C16, and C18 are myristic, palmitic, and stearic acids, respectively.

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Table 2.

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Degree of Modification and Residual Muramidase Activity of Lysozyme Acylated with Different Fatty Acids

Myristoylated

Palmitoylated

Stearoylated

Target

Modificationa (mol / mol)

Muramidase b Activity (%)





0

0.0

100

0.0

100

0.0

100

1

0.2

63

1.0

98

0.4

96

2

0.6

61

2.0

76

0.9

91

3

2.0

60

2.7

59

1.0

90

4

2.4

59

3.2

48

2.0

75

a

Determined by measuring the TNBS-non available _-amino groups The decrease of turbidity at 600 nm for 1 min of M. lysodeikticus cell wall suspension in K-phosphate buffer (pH 7.0) was monitored upon addition of lysozyme (1.8 µg / mL). The activity was represented as a percentage of that of the unmodified lysozyme. b

palmitoylated-lysine residues are located at the C-terminal of two helices in the upper cleft of the active site (Fig. 6). The two sites of incorporation were surface exposed.

THE MECHANISM OF ANTIMICROBIAL ACTION OF FATTY-ACYLATED LYSOZYME It is generally accepted that the competence for membrane fusion requires an accessible hydrophobic stretch to reside within the lipid bilayer when the protein penetrates into the membranes. In order to confirm the hypothesis that the palmitate residue directly mediated the interaction and penetration of lysozyme into the membrane and to prove our strategy, the ability of di-palmitoylated lysozyme (P2-Lz) to form pores in the liposomal membrane (lipid bilayer) was

tested. Liposomes loaded with K+ ions were made from E. coli-phospholipid and suspended in Na-phosphate buffer containing a positively charged fluorescence probe, 3,3'diisopropylthiodicarbocyanine (diS-C 3-(5)). Liposomes were hyperpolarized (negative inside) by valinomycin to allow fusion of the probe into the liposome (fluorescence quenching). The addition of P2-Lz (10 µg/ml) to the hyperpolarized liposomes reversed the fluorescence quenching, but the addition of native lysozyme (NLz) at the same concentration had no effect on the signal. The reversal of the fluorescence quenching by P2-Lz reflected its ability to disrupt the electrochemical potential which generated a rapid and significant collapse of the membrane potential similar to the addition of nigericin [13]. The results suggest that P2-Lz can dissipate the membrane electrochemical potential in E. coli liposome as a result of a specific or non-

Fig. (6). Stereographic representation of the three-dimensional structure of (P2-Lz) lysozyme illustrating the locations of the two palmitic acids (K33 and K97), and the two catalytic residues Asp52 (D52) and Glu35 (E35).


specific interaction. It is probable that the perturbation effect of P2-Lz on the membrane potential is due to an interaction with the CM through the attached hydrophobic palmitoyl residue. Therefore, the ability of fatty-acylated lysozyme to damage the OM and subsequent penetration into the CM may account for its bactericidal action against E. coli. These results also support the hypothesis that the killing site of Gram-negative bacteria would be the CM. In summary, our strategy to design an antimicrobial lysozyme for Gram-negative bacteria was based on lipophilization with a hydrocarbon chain so as to facilitate its fusion and penetration through the protective barrier. The results suggest that a hydrophobic peptide fused to lysozyme may promote the penetration of this cationic molecule into the CM of bacteria and the subsequent loss of the electrochemical membrane potential.

GENETIC-FUSION STRATEGY FOR ANTIMICROBIAL DESIGN The promising results with the fatty-acylated lysozymes prompted us to try a more precise approach for the design of a potent antimicrobial lysozyme using recombinant DNA technology. The simplest approach to build an amphiphilic lysozyme that mimics the structure of fusogenic proteins is to transfer hydrophobic domains to lysozyme. To mimic such a structure successfully, the design must not only transplant the hydrophobic domain to the molecule correctly, but must also exclude unfavorable interactions between the introduced hydrophobic domain and the rest of the protein,


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particularly the active site of the enzyme. Structure analysis indicated that fusion of hydrophobic peptides, which are able to form β-strand conformations of about the same length as myristic, palmitic, or stearic acid, to the C-terminus of lysozyme might be successful. The fact that fusion of a hydrophobic peptide to the C-terminus may guarantee the exposure of the domain to the exterior of the molecule, without interference with the active site, was suggested by the three-dimensional structure of lysozyme (Fig. 7).

CONSTRUCTION OF THE HYDROPHOBIC PEPTIDE-FUSED LYSOZYMES For this part, three oligonucleotide fragments were synthesized encoding the designed hydrophobic peptide sequences, as shown in Fig. 8: a tri-hydrophobic peptide, H3 (F-V-P); a penta-hydrophobic peptide, H5 (F-F-V-A-P); and a hepta-hydrophobic peptide, H7 (F-F-V-A-I-I-P). The cDNA sequence of lysozyme suggested an obvious way of fusing the hydrophobic peptides immediately after its Cterminus Leu-129. The cDNA of lysozyme was cloned and an appropriate cutting site (Pst I) was introduced by means of site-directed mutagenesis as previously described [12]. As outlined in Fig. 8, the mutant cDNA was digested with Pst I endonuclease followed by ligation, separately, with the synthesized double stranded DNA fragments encoding the codons of the respective hydrophobic peptide and two additional sequences (amino acids codons at the 5'- terminus that are lost by digestion and the stop codon at the 3'terminus). The full-length of the fusion cDNAs were confirmed by sequencing. First by isolation from the

Fig.(7). Stereographic representation of lysozyme illustrating the location of the genetically fused hydrophobic peptide to the Cterminus Leu 129.

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Ibrahim et al.

Fig. (8). Scheme for construction of the fusion gene of lysozyme. A Pst I site was first introduced at the C-terminal of the coding region by site-directed mutagenesis. Three double stranded DNA fragments, each encoding for the indicated hydrophobic amino acid sequence, were inserted into the Pst I site. In the cDNA: the dark box, the signal peptide sequence; the open box, the coding region; the bold line, the 3'-noncoding region.

cloning vector, and then by subcloning to the yeast expression vector pYG-100. These vectors were transformed to and expressed by Saccharomyces cerevisiae AH22. The wild-type (Wt-Lz) as well as the hydrophobic peptide-fused lysozymes were secreted into the medium and purified with two passes of cation-exchange chromatography (CMToyopearl 650) [13].

conformational changes and decreased muramidase activity in proportion to the length of the peptide. However, sufficient catalytic activity (necessary to hydrolyze the peptidoglycan of bacteria) was conserved in all of the fusion lysozymes (Table 3).

ANTIMICROBIAL ACTION LYSOZYMES AGAINST E. COLI

OF

FUSION

ENZYME ACTIVITY OF FUSION LYSOZYMES The enzyme activities at pH 5.5 of the fusion lysozymes were about 75-80% of that of the Wt-Lz [11], but were decreased at pH 6.0 (Table 3). The pH dependence of the enzyme activity (a shift to acidic pH) may be due to conformational changes. This is consistent with the fact that the hydrophobic domains of a protein tend to be exposed at an acidic pH, hence they will not interfere with the folding of the molecule. These results suggest that the conformation of lysozyme is slightly strained by fusion with the hydrophobic peptides, but without dramatic loss of enzymatic activity. This was confirmed by CD analysis of the fusion proteins [13] and by measuring their reactivity with monoclonal antibodies sensitive to conformational changes of lysozyme [11]. The results indicated that the fusion of the hydrophobic peptides to lysozyme causes

The hydrophobic fusion lysozymes showed significant bactericidal activities against E. coli compared to the Wt-Lz (Table 3). The bactericidal activities of the H5-Lz and H7-Lz constructs were the highest and similar in magnitude. This result also suggests that the antimicrobial action against E. coli increases with the increasing length of the hydrophobic peptide up to five residues. Furthermore, these results are consistent with the data for the fatty-acylated lysozymes suggesting that hydrophobic additions to lysozyme confers killing activity to Gram-negative bacteria. The sensitivity of E. coli to the hydrophobic peptide-fused lysozymes is best accounted by the amphiphilic nature of the molecule, which may contribute to the enhanced interaction with the complementary amphiphilic membrane lipid bilayer of bacteria.


Table 3.


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The Enzyme Activity, Antibacterial Action against E. coli , and Binding-activity to E. coli-LPS of Lysozyme as a Function of Fusing Hydrophobic Peptides of Different Lengths

Lysozyme

Muramidase activity (%) a

Anti-E. coli activity (cell Survival, %)b

LPS-binding activity (Residual activity, %) c

Wild-type (Wt-Lz)

100.0

94.6 ± 3.1

85.0 ± 2.6

Tripeptide-fused (H3-Lz)

81.1 ± 1.4

70.3 ± 1.8

78.8 ± 2.4

Pentapeptide-fused (H5-Lz)

62.2 ± 1.9

20.3 ± 0.9

56.3 ± 1.4

Heptapeptide-fused (H7-Lz)

52.7 ± 0.9

18.9 ± 0.7

53.8 ± 1.0

a The decrease of turbidity at 450 nm for 1 min of M. lysodeikticus cell wall suspension in Na-acetate buffer (pH 6.0) was monitored upon addition of lysozyme (1.8 µg / mL). The activity was represented as a percentage of that observed for Wt-Lz at pH 5.5. b Lysozyme (10 µg / mL) was incubated with bacteria (105 cells / mL) at 37 ˚C for 30 min in 50 mM Acetate buffer (pH 5.5), before determining survival on nutrient agar plates. c Binding-activity to LPS was assessed by incubating lysozyme (1 µg / mL) with thirty-fold weight excess (30 µg / mL) of LPS at 37 ˚C for 15 min in 50 mM acetate buffer (pH 5.5), then the residual enzyme activity was determined as above. The data are presented as a % of activity in the absence of LPS.

THE NOVEL ANTIMICROBIAL MECHANISM OF FUSION LYSOZYMES

DRUG DELIVERY-BASED ANTIMICROBIAL DESIGN

The interaction and fusion of the peptide-fused lysozymes with the LPS of Gram-negative bacteria was examined by monitoring the reduction in lysozyme activity of E. coli LPS vesicles (Table 3). A sharp decrease in the residual lysozyme activity was observed with the hydrophobic peptide-fused lysozymes (again up to the pentapeptide) compared to the Wt-Lz enzyme, indicating a strong interaction. We also examined the role of the catalytic activity of the engineered H5-Lz in the killing of E. coli by mutating the catalytic residue Glu-35 to Ala to inactivate the enzyme [11]. The inactivated H5-Lz showed only a slight anti-E. coli activity, indicating that the enzymatic activity of the modified lysozyme is important for complete expression of the antimicrobial action against Gram-negative bacteria. However, the largest factor seems to be the amphiphilic nature of H5-Lz, which most probably enables the molecule to insert itself into the lipid bilayer of the CM after reaching and hydrolyzing the peptidoglycan layer. In order to confirm this hypothesis the ability of H5-Lz to permeabilize the artificial liposomal membrane and E. coli ghost RSO vesicles (right side out) was investigated (for the detailed assay procedure refer to [13]).

Despite its very weak antimicrobial activity against Gram-negative bacteria, lysozyme is known to interact with LPS and subsequently distort the normal packing between phospholipids and LPS in the OM of bacteria [13, 36], as was also observed for Wt-Lz in Table 3. Further, studies performed in our laboratory indicated that lysozyme induced transient leakage of a periplasmic enzyme (alkaline phosphatase) and even a cytoplasmic enzyme (βgalactosidase) from E. coli [13]. The most probable explanation is that lysozyme with its catalytic function and ability to interact with LPS may be able to reach the CM but is not able to exert a lethal event, unless equipped with a membrane active domain such as a fatty acid or a hydrophobic peptide as discussed above. Here, the strategy is to deliver and localize a naturally occurring membraneactive aldehyde at the CM by coupling it to lysozyme.

The addition of H5-Lz to valinomycin hyperpolarized liposomes reversed the fluorescence quenching, but the addition of Wt-Lz at the same concentration had no effect on the signal, indicating the ability of H5-Lz to fuse into the membrane and induce collapse of the membrane potential similar to the effect of nigericin [13]. This result was confirmed using membrane vesicle derived from E. coli [39]. The pore-forming ability of H5-Lz was further evident by incubating the naturally sealed right side out (RSO) membrane vesicles of E. coli (RSO-vesicles) with H5-Lz before generation of the membrane potential by glucose addition. An obvious dissipation of the membrane electrochemical potential was observed, indicating that the mechanism by which H5-Lz kills Gram-negative bacteria is by promoting interaction with and insertion into the CM of the bacteria.

STRATEGY

FOR

One candidate aldehyde, commonly used as a food preservative, is perillaldehyde, the major constituent of the edible green leaves of Perilla frutescens [40]. It has long been used in traditional Chinese herb medicines. Perillaldehyde has moderate antimicrobial activity, but its application in food and drug applications is hampered for two reasons: (1) its strong odor and poor water solubility; and (2) its high hydrophobicity excludes it from the cell envelop of bacteria. The antimicrobial effect of perillaldehyde has been reported to be relatively more pronounced to Gram-negative bacteria than to Gram-positive ones [40]. On the other hand, lysozyme is more effective against Gram-positive bacteria. Hence, we postulated that lysozyme might act synergistically with perillaldehyde against both types of bacteria if they were bound together.

COUPLING PERILLALDEHYDE TO LYSOZYME To achieve a stable amide bond between perillaldehyde and the ε-amino lysyl groups of lysozyme, a technique to form a Schiff base was adopted [41]. As outlined in Fig. 9, lysozyme was incubated with varying mole ratios of

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Ibrahim et al.

Fig. (9). Schematic representation of the preparation for the covalent modification of lysozyme with perillaldehyde through Schiffbase formation. PA, perillaldehyde.

perillaldehyde under reducing condition in the cold [15]. Incubation of 3, 7, and 50 moles of perillaldehyde with lysozyme produced conjugates with 0.5 (PA1-Lz), 2.4 (PA2Lz), and 3.9 (PA4-Lz) mol of modified Lys residues, respectively, as judged by the TNBS method. It seems that only four Lys residues of lysozyme are readily reactive while the other residues may be buried in the interior of the molecule. Similar results were obtain in the fatty acylation experiments. This modification had a minimal effect on lysozyme solubility (92% of protein solubility was retained). Although it has been reported that interaction of aldehydes (hexanal) leads to extensive damage to the amino acids and polymerization of lysozyme [42], our perillaldehydelysozyme conjugates seem to be a promising compound because we found little structural changes by fluorescence analysis [15].

ENZYME ACTIVITY OF LYSOZYME CONJUGATES

PERILLALDEHYDE-

The muramidase activities of these derivatives, expressed as a percentage of the unmodified lysozyme, are shown in Table 4. The decrease in lysozyme activity is dependent on the degree of modification. However, high residual activity

can be preserved even with up to four aldehyde residues per molecule.

THE NOVEL ANTIMICROBIAL ACTIVITY OF PERILLALDEHYDE-LYSOZYME CONJUGATES The antimicrobial assay of lysozyme modified to various degrees with perillaldehyde employed Escherichia coli K12, as the Gram-negative bacteria, and Staphylococcus aureus as the Gram-positive bacteria (Table 4). Modified lysozymes had significant bactericidal activity against both microorganisms, and the killing potency was greatly intensified with an increase in the degree of modification. This enhanced antimicrobial activity was not observed with the unreacted mixtures of perillaldehyde and lysozyme at mole ratios (PA:Lz, 2:1 and 5:1) almost similar to those in their conjugates (PA2-Lz and PA5-Lz). In addition perillaldehyde alone had less antimicrobial effect when tested at a 5 times greater concentration than lysozyme. The antimicrobial effects of the conjugates were stronger against S. aureus than against E. coli. These results provide direct evidence that attachment of perillaldehyde to lysozyme made the protein active against Gram-negative bacteria at very low concentration and improved its antimicrobial effect against the partially resistant Gram-positive bacteria. Furthermore, it can also be concluded that both compounds involved in the conjugate are directly involved in the novel killing action,



683

Table 4. The Enzyme Activity and Antibacterial Action against E. coli and S. aureus of Lysozyme Modified to Various Degrees with Perillaldehyde

Muramidase activity

Anti-E. coli activity

Anti-S. aureus activity

Lysozyme (%) a

CFU / mL

(Survival, %)b

CFU / mL

(Survival, %)b

Native

100.0

0.22 x 105

100.0

2.1 x 103

100.0

PA1-Lz

94.9 ± 0.4

1.80 x 104

83.1 ± 1.8

9.0 x 102

42.1 ± 2.4

PA2-Lz

89.4 ± 0.9

5.90 x 103

27.0 ± 1.1

6.9 x 102

32.2 ± 1.6

PA4-Lz

73.3 ± 0.7

4.40 x 103

20.2 ± 1.0

0.2 x 102

9.4 ± 0.4

Mix 2:1c

ND

0.26 x 105

120.2 ± 3.0

1.7 x 103

77.2 ± 3.7

Mix 5:1

ND

0.25 x 105

114.6 ± 2.8

2.5 x 103

115.8 ± 2.9

a The decrease of turbidity at 450 nm for 1 min of M. lysodeikticus cell wall suspension in Na-phosphate buffer (pH 6.2) was monitored upon addition of lysozyme (0.85 µg / mL). The activity was represented as a percentage of that observed for N-Lz. ND; not determined. b Lysozyme (100 µg / mL) was incubated with bacteria (105 cells / mL) at 37 ˚C for 60 min in 10 mM K-phosphate buffer (pH 7.0), before determining the colony forming unit (CFU) on agar plates. The data are also represented as percentage survival to that of unmodified lysozyme. c Mix 2:1 and 5:1 indicate mixtures of aldehyde and lysozyme at mole ratios of 2 and 5, respectively.

but they could not operate synergistically when they were mixed without being conjugated.

GENERAL MECHANISM OF ANTIMICROBIAL ACTION OF NEW LYSOZYMES Although the different approaches to design antimicrobial function discussed in this review introduce a new conceptual use of lysozyme that extended its narrow antibacterial spectrum, all approaches appear to share a common mechanistic framework, outlined in Fig. 10. All of the modified enzymes targeted the killing site of bacteria, generally the CM. Lysozyme, a polycationic molecule, with characteristic amphiphilic nature, may be associated with and disrupt the OM by distorting the normal packing between phospholipids and LPS [18]. As a consequence, the enzyme may pass through the bacterial envelope and approach the site of enzymatic action (peptidoglycan) while residing in the aqueous periplasm. It is generally accepted that competence in the membrane fusion process corresponds to the flexible folding of a protein while an accessible hydrophobic stretch must reside within the lipid bilayer when the protein penetrates into the membranes. The hydrophobically-tailed lysozyme molecules (fatty-acylated or genetically fused with hydrophobic peptides) disrupt the function of the CM by membrane fusion. They do this by burying their hydrophobic extensions into the lipid bilayer while positioning the positively charged residues of lysozyme near the negatively charged phosphate groups of the bilayer to form pores into the lipid bilayer. This is supported by the membrane permeabilization experiments. For the hydrophobic perillaldehyde-lysozyme the mechanism seems to be insertion into the two planes of the CM bilayer to interfere with various biosynthetic processes, such as inhibition of transport of nutrients and macromolecule precursors, leading to cell death.

DESIGN OF ANTIMICROBIAL PEPTIDES DERIVED FROM LYSOZYME AND APROTININ Bactericidal Properties of Denaturated Lysozyme and Aprotinin Since its discovery it has been believed that the only mechanism through lysozyme explicates its bactericidal activity consists in the enzymatic degradation of the glycosidic β-linkage between N-acetylhexosamines of the peptidoglycan molecule present in the bacterial cell wall. This idea was put in question for the first time only recently in 1985 when Laible and Germain were able to show that denaturated lysozyme, enzymatically inactive, was as effective as the native molecule against two streptococcus strains namely Streptococcus sanguis and Streptococcus faecalis [43]. The result of Laible and Germain, indicating for lysozyme a mechanism of action independent from its enzymatic activity, was latter confirmed from several other investigators which could show that denaturated lysozyme deprived of enzymatic activity was able to kill efficiently E. coli [5,6,44] suggesting that the bactericidal potency of lysozyme is not only due to its muramidase activity but also to its cationic and hydrophobic properties. Aprotinin, a protease inhibitor, shows a similar behaviour. In fact the denaturation and precipitation of aprotinin with DTT, which reduce the intramolecular disulfide bonds, yields a compound devoid of any protease inhibiting activity which still retains its bactericidal potency [5] which indicate, as already observed for lysozyme, that the bactericidal activity of aprotinin is probably related to its cationic and hydrophobic character rather than to its activity as protease inhibitor. Similarly to lysozyme and aprotinin, also cathepsin G, a proteolytic enzyme, present in human neutrophil granules, with bactericidal properties retains its bactericidal properties after the destruction of its catalytic activity [45,46] (see also the review of Shafer and co-workes

684


Ibrahim et al.

Fig. (10). Schematic representation of the membrane structure of Gram-negative bacteria illustrating the possible mechanism by which engineered lysozymes kill the bacteria. The lysozyme molecule is represented as a stippled ball (Lz) and the attached domain (can be either a hydrophobic stretch or hydrophobic perillaldehyde) as a black extension. The vertical arrow represents permeabilization of membrane.

in this issue) . At present, cause a limited number of reports, it is not clear whether the observed events, that antimicrobial properties of proteins are independent of their biochemical activities, are a part of a general phenomenon or whether this is limited to a few proteins.

sequence of lysozyme from residue 98 to 112 [16] (peptide P 2) Although the bactericidal activity of the peptide P 2 (Table 5) was considerably less bactericidal than lysozyme its bactericidal properties were considered as a good basis to develop peptides with higher bactericidal activity.

BACTERICIDAL LYSOZYME

FROM

DESIGN BACTERICIDAL PEPTIDES FROM THE PEPTIDE P2

The presented evidence that lysozyme is able to explicate its bactericidal action independently from its enzymatic activity, making it essential to determine whether the antimicrobial action of lysozyme requires the whole molecule or just smaller peptide within its sequences can fulfill this function. To answer this question, lysozyme was proteolytically digested with clostripain, a protesase from Clostridium bacteria which are present in the intestinal micro flora. Trypsin-like activity is characteristic of clostripain which specifically cuts the peptide bond after arginine residue thus generating a number of peptides with C-terminus arginine. The presence of a positively charged amino acid like arginine in the C-terminus of the polypeptide chain is considered an important factor for the antibacterial activity of the peptides. Lysozyme digested by clostripain yielded a bactericidal peptide fragment of (15 residues) whose amino acid sequence corresponds to the

The strategy followed to develop more potent bactericidal peptides starting from the amino acid sequence of P2 consisted as a first step to synthesized analogous peptides that differ from the parent peptide, P2, at one sequence position and then, latter, research for more short sequences with bactericidal properties.

PEPTIDES

DERIVED

THE IMPORTANCE OF THE PRESENCE OF CERTAIN AMINO ACIDS IN THE POLYPEPTIDE CHAIN FOR THE BACTERICIDAL ACTIVITY This approach consists to determine which residues are important for the antimicrobial activity of the peptide. To this purpose two analogous peptides of the peptide P2 were synthesized. In one the tryptophane residue in position 108 was replaced with the similar but less hydrophobic amino



acid tyrosin whereas in the other, tyrosin replaced the tryptophane residues in the position 111 (Table 5). This change in the amino acid composition yielded the lost of the most of the antibacterial activity of the original peptide, P2, indicating on one side the importance of the presence of the tryptophane residues, in the position 108 and 111, for the bactericidal properties of P2 and on the other side showing that hydrophobic character of the peptide is a significant element for its bactericidal activity. Another aspect of the bactericidal properties of the peptide to be investigated concerns to examine the importance of the cationic character. Replacement of Asn 106 with the positively charged arginin strongly increases the antibacterial activity of the peptide Table 5.

685

suggesting that to increase the positive charge of the peptide is a successful strategy to improve the bactericidal properties of the peptides. It is to worthy to note that the mutation Asn 106 to Arg 106 occurred naturally in the human and baboon lysozyme.

DETERMINATION WHICH PART OF THE BACTERICIDAL MOLECULE IS IMPORTANT FOR BACTERICIDAL ACTIVITY The peptide P 2 is an amphipathic molecule in which the polar part is represented from the N-terminal sequence 98-

Antibacterial Activity of Lysozyme and the Sythetic Analgue Peptides Derived From its Bactericidal Domain Activity * Organisms

Strain or type

P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

P11

Escherichia coli

ATCC25922

2.0

0.3

1.0

0

0

0

0.2

1.7

2.0

0

2.0

Klebsiella Pneumonieae

ATCC 13883

1.7

0.2

0.1

0

0

0

0

1.3

1.0

0

0.6

Pseudomonae aercescens

WS

1.0

0

2.5

0.2

0

0

0

2.7

2.7

0.3

0.7

Serratia marcescens

ATCC 8100

1.6

0.1

0.4

0

0

0

0

2.0

0.1

0

0.6

Bacillus subtilis

BGA

2.6

2.0

2.0

0.2

0

0

0.4

2.0

2.3

0

2.3

Micrococcus lueus

ATCC4698

2.5

0

1.5

0

0

0

0

3.0

1.4

0

1.7

Staphylococcus aureus

ATCC 259230

0.2

0.1

1.6

0

0

0

0

3.0

0.4

0

1.0

Staphylococcus epidermidis

ATCC12228

0.2

0

0.6

0

0

0

0

2.9

1.6

0

1.2

Staphylococcus lentus

WS

2.7

0

2.0

0

0

0

0.4

2.6

1.6

0.1

2.7

Streptococcus zoepidemicus

WS

0.7

0.2

0.6

0

0

0

0

2.5

2.5

0.2

2.0

Candida albicans

ATCC2091

2.4

0

0.3

0

0

0

0

3.0

3.0

0

3.0

P1Chicken lysozyme P2 I - V - S -

D -

G -

N -

G -

M-

N -

A -

W-

V -

A -

W-

R

Peptide 98-112 of chicken lysozyme

P3

I -

V -

S -

D -

G -

N -

G -

M-

N -

A -

W-

V -

A -

W-

R

P4

I -

V -

S -

D -

G -

N -

G -

M-

N -

A -

W-

V -

A -

W-

R

Arginin replaces asparagin (corresponding to human lysozyme peptide) Thyrosi9n replaces trypthophane 108

P5

I -

V -

S -

D -

G -

N -

G -

M-

N -

A -

W-

V -

A -

W-

R

Thyrosin replaces trypthophane 111

P6

I -

V -

S -

D -

G -

N -

G -

M

N-terminat “moiety” (octapeptide)

P7

N -

A -

W-

V -

A -

W-

R

C-terminat “moiety” (heptapeptide)

P8

R -

A -

W-

V -

A -

W-

R

Arginin replaces asparagin

R -

A -

W-

V -

A -

W-

R

D-enantiomer of the substituted C-terminal heptapeptide

P10

A -

W-

V -

A -

W-

R

C-terminal hexapeptide

P11

A -

W-

V -

A -

W-

R

Arginin replaces alanin n the hexapeptide

101 102 103 104 105 106 107 108 109

110

P9

(D)

98 99 100

111

112 N-termC-term

Antibacterial activity is shown as log N0 /N1 where N0 refers to the control number of colonies without antibacterial material and N1 refersa to the number of colonies containing antibacterial agent after 2 hours incubation. Bacterial which were lysed by chicken lysozyme. The chicken lysozyme and the oligopeptide were assyed of a concentration of 8.3 x 10-10 and 25 x 10 -9 mol/assay respectively. The boldface aminbo-acids have been replace4d in the polypeptide sequence I-V-S-D-G-N-G-M-N-A-W-V-A-W-R. Reprinted from A. Pellegrini et.al. J. of Appl. Bacteriol. 82,372-378 (1997) with permission from Blackwell Science Ltd.

686


105 whereas the hydrophobic part is represented from the peptide 106-112. Moreover the peptide P2 is a part of a helix–loop-helix (α helical hairpin) domain of chicken egg white lysozyme (residues 88-114) in which the residues 98105 are contained within the N-terminal helix and loop structure whereas the residues 105-112 are entirely present in the C-terminal helix (Fig. 11). Two peptides with the amino acid sequences corresponding to the 98-105 and 106-112 were synthesized and their bactericidal properties investigated. It could be shown that the peptide 98-105 was devoid of any bactericidal activity whereas the peptide 106112 retained part of the bactericidal activity of the parent peptide 98-112 indicating on one side that the presence of both helixes is necessary to develop an optimal antibacterial activity and on the other side that a partial activity is retained from a single helix structure.

DESIGN OF SHORT BACTERICIDAL PEPTIDES Once determined which part of the amino acid sequence of P2 retains the most of the bactericidal activity, it was though to design short bactericidal peptides following the procedure just above described which consists to replace one of the amino acids of the peptidic sequence with another one possessing a positive charge. Following this criterion Asn 106, of the peptide 106-112, was replaced with arginine. The synthetic peptide , P8, was strongly active against all the bacteria strains investigated. After this positive result it was investigated the bactericidal properties of two shorter peptides. One peptide consisted of P8 deprived of the Nterminal Arg. This peptide, P10, presented a very weak bactericidal activity against a limited number of bacteria. which confirms the importance of Arg in the polypeptide chain for bactericidal activity. The importance of arginine in the polypeptide chain was furthmore confirmed by the results of the investigation of the bactericidal properties of

Ibrahim et al.

the synthetic peptide P 11 which consisted of P8 deprived of Ala 107. Peptide, P11, was as bactericidal as P8. The effect of the stereospecific complementarity between peptide and bacteria was also investigated by the synthetic Denantiomeric peptide P8. This peptide was slightly weaker than the peptide P 8 indicating that the mechanism of action of the bactericidal peptides do not involve stereospecific interaction with receptors of the bacterial membrane. Similar results were obtained with cecropin-melittin hybrids [48,49]. The non stereospecific interaction with bacteria membrane receptor is not however a general rule. In fact D-enantiomer of apidacin , a bactericidal peptide isolated from honeybee, is completely devoid of antibacterial activities [50]. APROTININ Aprotinin is one of the protease inhibitor most extensively investigated . It was discovered by Kraut and coworkers in 1928 [51] as a kallikrein inhibitor present in human blood. Some years later Kunitz and Northrop [52] were able to isolate it as a trypsin inhibitor from bovine pancreas and in 1950 Astrup [53] found it in the bovine lung . The fact that it is present in several bovine organs and that it inhibits several proteases [54,55] and that it was isolated from several researchers at different time creates some confusion about its name. In fact aprotinin is also known under several synonyms such as bovine pancreatic trypsin inhibitor, kallikrein inhibitor, Kunitz proteinase inhibitor and it is available as a drug under the name Trasylol. Therapeutically aprotinin is used as kallikrein inhibitor in patients suffering from acute pancreatitis and as hemostatic to reduce the blood loss and consequently to limit the need of blood transfusion in patients undergoing cardiac surgery [56,57] Aprotinin is a small basic protein ( pI 10.5) of 6.5 KD composed of a single polypeptide chain of 58 amino acid

Fig. (11). Schematic ribbon representation of α-helical hairpin motif of hen egg lysozyme. The secondary structural elements are: αhelix 1, Asp 87-Ser100; α-helix 2, Ala107-Arg114; and loop Asp101-Asn106. The tightness of the loop between the two helices is constrained by Gly 102 and Gly 104.



residues cross-linked by three disulfide bridges. Selective cleavage of only one disulfide bridge do not influence the inhibitory activity of the protein [29]. Although isolated already in 1930 and extensively studied since then, the biological function of aprotinin remains unknown [54]. Aprotinin also has antimicrobial property. It is able to inhibit the bacterial growth of several gram-negative and gram-positive bacteria in vitro and in vivo [59] and the replication of influenza virus [60,61]. Antibacterial and antiviral properties of aprotinin have been attributed to its antiproteinase capacity. However a wide variety of proteinase inhibitors have no antibacterial properties [32].

ANTIVIRAL APROTININ

PEPTIDE

DERIVED

FROM

The results of our earlier investigations showed that the bactericidal properties of aprotinin are not necessarily ascribed to its protease inhibitory function. Thus it was considered of great interest to investigate whether some internal amino acid sequence of the aprotinin retained all or partially the bactericidal properties of the whole molecule. For the reasons just above discussed for lysozyme the proteolytical digestion of aprotinin was performed by clostripain. Proteolytical digestion of aprotinin with clostripain yielded three bactericidal peptides [62] and an antivirale one [63] (Fig 12). The amino acid sequence of the antiviral peptide, YFYNAK, corresponds to the sequence 2126 of the intact aprotinin . It inhibited the replication of herpes simplex virus 1 in rhabdomyosarckom cells in a dose-dependent manner . The 50% effective dose was 38 µ M. This hexapeptide, is devoid of any antiproteolytic and antibacterial activity, exerted antiviral activity against the HSV-1 and PI3. Its sequence does not correspond to the amino acid sequence of the active site of aprotinin (amino acid sequence 13-18) , thus it appears that there is not any apparent relationship between antiviral activity and proteinase inhibition. Although it was previously reported that aprotinin inhibits influenza virus replication [61] it had no effect on the multiplication of the HSV-1 and PI3. The fact that in the amino acid sequence of aprotinin is present the sequence of a small peptide with antiviral activity resembles only partially to the situation of cystatin, a

687

cysteine protease inhibitor. The sequence of a tripeptide derived from the amino acid sequence of the active site of cystatin [45] is able to inhibit the replication of the herpes simplex virus 1 [64]. The short peptide derived from cystatin possesses antiprotease activity thus in that system one can suppose that it can interfere with some viral cysteine protease(s) crucial for the virus replication.

BACTERICIDAL APROTININ

PEPTIDES

DERIVED

FROM

The three bactericidal peptides derived from the clostripain digestion of aprotinin cover, with the exception of the dipeptide Ala16-Arg17, the whole amino acid sequence of aprotinin molecule (Fig. 12). All three peptides did not show any antiproteolytical activity. The most active peptide was P 18-39 which exerts a strong bactericidal activity against a wide spectrum of Gram-positive and Gram-negative bacteria (Table 6). Moreover its bactericidal activity was higher than its parent molecule. In comparison with the other peptides it has the most marked hydrophobic and basic characters which are considered important for bactericidal activity. Bacteria strains like S. aureus and S. epidermidis which were not sensitive to the action of aprotinin were highly susceptible to P18-39 . The peptide P1-15 is an acidic peptide (pI 6.18). Its bactericidal activity was directed essentially against Gram-positive bacteria. We have recently observed a similar phenomenon. Anionic peptides derived from the proteolytical digestion of the proteins αlactalbumin and β-lactoglobulin were bactericidal against Gram-positive bacteria strains only[65,66]. These results indicate that bactericidal properties are not a prerogative of the only basic peptides but the acidic peptides can explicate this function as well. Recently the bactericidal properties of several anionic peptides have been confirmed from others [67,68]. The bactericidal activity of P1-15 against grampositive bacteria was higher in comparison to that of aprotinin, this latter was however higher than that of P1-15 against Gram-negative bacteria. The third peptide derived from the proteolytical digestion of aprotinin, P 40-58 , showed a narrow spectrum of bactericidal activity. Only B. brochiseptica, S. marcescens, B. subtilis and S. lentus were sensitive, at different extent, to the action of P40-58 .

Fig. (12). Amino acid sequence of aprotinin and of the three bactericidal peptide fragments derived from the proteolytical digestion of aprotinin by clostripain. The peptide fragment YFYNAK presented antiviral activity against HSV-1 and PI3.

688


Table 6.

Ibrahim et al.

Antibacterial Activity of Aprotinin and the Aprotinin Fragments Activity* Organisms

Strain or type

Peptide 1-15 7,8x10 -9 mol/assay

Peptide 18-39 7x10 -9 mol/assay

Peptide 40-58 7x10 -9 mol/assay

Aprotinin 7x10-9 mol/assay

Brodetella bronchiseptica

ATB 32 GN

0.1

1.8

0.4

0.5

Escherichia coli

ATCC 25922

0.2

2.6

0.0

0.8

Klebsiella pneumoniae

ATCC 13883

0.0

3.5

0.0

0.3

Pseudomonas aeruginosae

WS

0.0

3.0

0.0

0.8

Serratia marcescens

ATCC 8100

0.1

1.1

1.0

0.8

Bacillus subtilis

BGA

0.6

3.0

0.1

1.0

Micrococcus luteus

ATCC 4698

0.2

2.3

0.0

0.0

Staphylococcus aureus

ATCC 259230

0.5

2.7

0.0

0.0

Staphylococcus epidermidis

ATCC 12228

2.0

3.4

0.0

0.7

Staphylococcus lentus

WS

3.2

2.0

0.9

3.5

Streptococcus zooepidemicus

WS

2.1

3.5

0.0

0.0

Candida albicans

ATCC 2091

0.0

3.0

0.0

0.0

*Antibacterial activity is shown as log (N0/N1) where N0 refers to the control number of colonies without antibacterial material and N1 refers to the number of colonies containing antibacterial agent after an incubation period of 2h Reprinted from A Pellegrini et al. Biochem. Biophys. Res. Commun., 222, 559-565 (1996) with permission from Academic Press

DESIGN BACTERICIDAL PEPTIDES FROM P18-39 OF APROTININ

BACTERICIDAL ACTIVITY IS PRESENT IN THE N-TERMINAL SHEET ONLY (TABLE 7)

Among the bactericidal peptides derived from the proteolytical digestion of aprotinin of particular interest is the peptide P18-39 not only because it possesses the higher bactericidal activity but also for its structural characteristic within the aprotinin molecule. Inside aprotinin structure P 1839 forms an antiparallel β sheet conformation connected by a short turn (β sheet hairpin) (Fig. 13). β sheet hairpin structures are known motifs present in several bactericidal peptides like defensins [69], the human bactericidal protein B/PI [70]and tachyplesin [71].

This structural situation resembles to the just above discussed situation concerning the bactericidal peptides derived from lysozyme. In both cases a complex biologic structure , α helix hairpin or β sheet hairpin , can be resolved to its single components and only one of them possesses biological activity. However both investigations show that intact original structure is important to explicate a higher bactericidal activity.

DETERMINATION OF WHICH PART OF THE SEQUENCE OF P18-39 IS IMPORTANT FOR BACTERICIDAL ACTIVITY As just above described P18-39 is structurally composed of two β sheets connected by a short loop, thus it came natural to investigate which part of the β sheets is important to develop a bactericidal activity. Two peptides P18-26 and P 29-39 representing everyone the single β sheet of P18-39 were synthesized and their bactericidal properties investigated [17]. Although Lys 26 does not really belong to the N-terminl sheet, it was considered important to include this amino acid in the peptide sequence which corresponds to the N-terminal sheet because positively charged , a characteristic this, which , as above mentioned, is important for bactericidal activity.

THE IMPORTANCE OF THE CATIONIC AND HYDROPHOBIC CHARACTER FOR THE BACTERICIDAL PROPERTIES OF P18-26 Once determined which part of the P18-39 is important for bactericidal activity the next investigative step consisted to design a peptide similar to P18-26 in which the C-terminal lysine was changed with a more positively charged arginine. This strategy was suggested by previous studies where arginine was found particularly important for bactericidal activity [16,47]. The peptide P*18-26 was more potent than P 18-26 particularly against Gram-negative bacteria, however when such a replacement was performed with P20-26 the already weak activity of this peptide was abolished , thus the only presence of arginine as the C-terminal amino acid does not explain adequately the bactericidal property of P*18-26 .


A


689

B

Fig. (13). Schematic ribbon representation of aprotinin (a) and of its bactericidal peptide fragment P18-39 (b). The secondary structural elements are : β-sheet 1, Arg 20-Ala 25; β-sheet 2, Leu 29-Gly 36 and loop Lys26-Gly28. The amino acid residues are represented in the figure by one letter symbol.

Having investigated the importance of the charge for the bactericidal activity of the peptides it was studied the effect of the hydrophobic character for the bactericidal properties of the peptides. Removing the N-terminal hydrophobic residues Ile-Ile of P*18-26 the bactericidal activity of the peptide was completely lost. A strong reduction of the bactericidal activity was also observed when the same deletion was performed on P18-26 . The importance of the hydrophobic character for the bactericidal activity of the peptides was confirmed when the bactericidal properties of the peptides P 18-26 FFVAP and P* 18-26 FFVAP, in which to their C-terminal parts was added the hydrophobic sequence FFVAP, was investigated (Table 7) . The importance, of the presence of this hydrophobic pentapeptide FFVAP into the peptide sequence, for the bactericidal activity was previously showed by Ibrahim and co-workers [13] (see also above). The bactericidal potency of the peptide P18-26 FFVAP and of the peptide P*18-26 FFVAP was comparable with that of the parent peptide, P 18-39 confirming that hydrophobicity is a crucial factor for the bactericidal activity of the peptides.

RETRO AND RANDOM PEPTIDE P18-26

ANALOGS

OF

THE

The importance of the bond direction and of the sequence of the peptide were investigated by mean of the synthetic retropeptide of P*18-26 , that is P*26-18 , and P*18-26 random. The position of the single amino acid in the polypeptide chains and the direction of the peptide bond seem not to play a crucial role. Retropeptide P*26-18 was just slightly less active than P*18-26 and there were only a minor difference between the bactericidal activity of the random peptide P*18-26 and that of P*18-26 . These results confirm those of other authors on the bactericidal properties of cecropin [49].

BACTERICIDAL MECHANISM OF THE PEPTIDES DERIVED FROM LYSOZYME AND APROTININ Investigations on the bactericidal mechanism of the antimicrobial peptides designed from lysozyme and

690


Ibrahim et al.

Table 7. Antibacterial Activity of Aprotinin and the Synthetic Analogue Polypeptides Derived from its Bactericidal Domain P18-39 P* 18-26 # P* 18-26 random FFVAP

#

P18-26 FFVAP

Organisms

Strain or type

P1-58

P18-39

P18-28

P29-39

P18-26

P* 18-26

P20-26

P* 20-26

P* 26-18

E. coli

ATCC 25922

0.2

2.6

0.0

0.0

0.3

1.4

0.3

0.0

1.6

0.6

1.3

1.1

B. bronchiseptica

WS

0.1

1.8

0.4

0.0

0.2

1.1

0.2

0.0

0.6

1.0

1.8

1.2

K. pneumonieae

ATCC 13883

0.0

3.5

0.2

0.0

0.0

0.1

0.0

0.0

0.6

0.9

1.9

1.2

P. aeruginosa

ATCC 27853

0.0

3.0

0.1

0.3

0.0

1.7

0.0

0.0

1.0

0.6

1.4

0.6

S. marcescens

ATCC 8100

0.1

1.1

0.2

n.d.

0.4

0.0

0.0

0.0

0.2

0.1

0.6

0.0

B. subtilis

BGA

0.6

3.0

1.3

0.0

1.5

>3.0

0.0

0.0

>3.0

>3.0

>3.0

>3.0

M. luteus

ATCC 4698

0.2

2.3

1.5

0.0

>3.0

>3.0

0.0

0.0

0.1

2.1

>3.0

>3.0

S. aureus

ATCC 259230

0.5

2.7

1.4

0.0

0.0

1.3

0.0

0.0

0.5

2.0

2.0

1.6

S. epidermides

ATCC 12228

2.0

3.4

0.2

0.0

0.9

2.1

0.0

0.0

1.4

3.0

3.0

2.0

S. lentus

WS

3.0

2.0

1.6

0.0

1.2

>3.0

0.1

0.0

0.5

1.9

>3.0

>3.0

Str. zooepidemicus

WS

2.1

3.5

1.0

0.0

2.9

1.0

0.1

0.0

0.7

1.9

>3.0

1.4

Candida albicans

ATCC 2090

0.0

3.0

0.7

0.0

0.2

0.2

0.3

0.0

0.0

2.5

2.3

1.9

P1-58, P18-39, P18-28, P29-39, P18-26, P* 18-26, P20-26, P* 20-26, P* 18-26 FFVAP, P18-26 FFVAP, P* 26-18, P* 18-26 random,

Aprotinin I-I-R-Y-F-Y-N-A-K-A-G-L-C-Q-T-F-V-Y-G-G-C-R I-I-R-Y-F-Y-N-A-K-A-G L-C-Q-T-F-V-Y-G-G-C-R I-I-R-Y-F-Y-N-A-K I-I-R-Y-F-Y-N-A-R R-Y-F-Y-N-A-K R-Y-F-Y-N-A-R I-I-R-Y-F-Y-N-A-R-F-F-V-A-P I-I-R-Y-F-Y-N-A-K-F-F-V-A-P R-A-N-Y-F-Y-R-I-I I-A-N-R-I-Y-R-Y-F

Peptide 18-39 of aprotinin N-terminal “moiety” of P18-39 C-terminal “moiety” of P 18-39 N-terminal “moiety” of P18-39 (nonapeptide) Arginine replaces lysine in P18-26 C-terminal heptapeptide of P18-26 Arginine replaces lsine in P20-26 Peptide, P* 18-26 , with the hydrophobic sequence, FFVAP, attached Peptide, P18-26 , with the hydrophobic sequence, FFVAP, attached Retropeptide of P 18-26 Peptide with the random sequence of P* 18-26

*Antibacteril activity is shown as log (N0N1) where N0 refers to the control number of colonies without antibacterial material and n1 refers to the number of colonies containing antibacterial agent after an incubation period of 2h. Polypeptides were assayed at a concentration of 7.5 x 10-8 mole per assay #The polypeptides with the attached hydrophobic sequence, FFVAP, were assayed at a concentration of 1.25 x 10-8 mol per assay because of their poor solubility WS: wild srain n.d.: not determinated Repeinted from A. Pellegrini et al. Biochem. Biophys. Acta 1433, 122-131 (1999) Copyright 1999 with permission from Elsevier

aprotinin fragments were performed with E. coli ML-35. A bacterial strain particularly suitable to analyse the interaction between bactericidal peptides and Gram-negative bacteria [72]. All the peptides investigated were able to increase the outer and the inner permeabilization of E. coli causing the bacteria death.

CONCLUDING REMARKS The strategies presented in this review clearly envisage that biochemical and genetic modifications are attractive means by which to design proteins with novel properties and new activities such as superior antimicrobial action. For example, the antimicrobial action of lysozyme can be improved to include resistant bacteria using three different strategies. The designs impart combinations of existing

bactericidal elements that may be difficult for microorganisms to overcome. Therefore, these approaches herald great opportunities for their potential use in development of safe foods and drugs. For food and drug technologists, the present data also permit us to emphasize that a safe food protein can be tailored to achieve a particular biotechnological function by transplanting a proper functional domain to the existing architecture of protein. Thus, further exploitations of these strategies with lysozyme and other biologically active molecules should provide valuable new tools for food and drug technologists. In this review a strategy has been presented for the rational design of shorter peptides. Such strategy is important because almost all of the bactericidal peptides are large molecule. The large size of the peptides represents a limitation in view of a therapeutic use because of possible hypersensitivity reactions. Moreover large peptides are expensive to produce,



thus an eventual loss of the bactericidal activity of the shorter peptide will be balanced by this positive factors. Eventually, the successful development presented in this review would add a new antimicrobial molecules to the decreasing arsenal of antibiotics available for the treatment of infections by resistant microorganisms.

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This work was supported by a Scientific Research Grant (C-2 #13660129) from the Ministry of Education, Science, Sports and Culture of Japan.

LIST OF ABBREVIATIONS CFU

= Colony forming units

CM

= Cytoplasmic membrane

DTT

= Dithiothreitol

RSOvesicles

Lysozyme derivative modified with perillaldehyde.

= Sealed Right-Side-Out ghost vesicles of E. coli

S-Lz

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Wt-Lz

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