Enhancement of antibacterial and lipopolysaccharide binding activities of a human lactoferrin peptide fragment by the addition of acyl chain

Andreja Majerle, Jurka Kidric and Roman Jerala*

Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia

Received 22 October 2002; returned 13 December 2002; revised 17 February 2003; accepted 18 February 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cationic antibacterial peptides are potentially therapeutic in the treatment of sepsis, because of their amalgamated antibacterial and lipopolysaccharide-binding activities. We prepared acyl analogues of the peptide fragment of human lactoferrin, which originally had weak antibacterial activity. It was found that 12 carbon units constitute the optimal acyl chain length, enhancing the antibacterial activity and binding of lipopolysaccharide by up to two orders of magnitude. Lactoferrin-based lipopeptides approached the activity of polymyxin B, a lipopeptide of natural origin, but were also active against Gram-positive bacteria.

Keywords: antibacterial peptide, endotoxin, human lactoferrin, lipopeptide


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and plants produce cationic antibacterial peptides as a first line of defence against invading pathogenic microorganisms.1,2 The structures of these peptides are highly diverse and can form different types of secondary structure, but they are all amphipathic and have a net positive charge under physiological conditions.3 The primary target of their action is bacterial membrane.4 Its leaflet contains, in contrast to multicellular animals, a large excess of anionic phospholipids. Development of resistance against such peptides, by modification of the membrane composition, is unlikely without compromising the bacterial viability. Antibacterial peptides may thus provide an alternative to conventional antibiotics, which are becoming increasingly ineffective due to the rapid emergence of resistant bacterial strains.

A major constituent of the cell wall of Gram-negative bacteria, lipopolysaccharide (LPS), is one of the most potent stimulants of the immune response and can be released from bacteria on administration of antibiotics.5,6 Following cellular recognition of LPS, inflammatory mediators such as cytokines, adhesion molecules and others are produced7 and may lead to septic shock.6,8 LPS comprises a lipid A moiety, which is the minimal structural element necessary for endotoxic activity.6,9 Attempts to develop molecules that prevent LPS binding to cellular receptors have often focused on the lipid A-binding region from endogenous LPS-binding peptides and proteins. The positive charge and hydrophobicity of peptides seem to be important in determining their ability to bind LPS. One of the most studied antimicrobial peptides is cyclic lipopeptide polymyxin B, which is effective against Gram-negative bacteria.10 Its medical use, however, is limited by its toxic side effects.11

Lactoferrin is an 80 kDa iron-binding glycoprotein found in exocrine secretions of mammals and in granules of neutrophils during inflammatory responses.12 It has antibacterial activity against a broad range of Gram-positive and Gram-negative bacteria and fungi.1315 Human lactoferrin binds to lipid A with high affinity16 and induces LPS release from the cell wall of Gram-negative bacteria.17 Proteolytic digestion of human lactoferrin yields a peptide fragment called lactoferricin H, which has enhanced antibacterial activity compared to intact lactoferrin.18 Lactoferricin contains a region that forms an amphipathic {alpha}-helix (residues 21–31), distinct from the site of iron binding19 and includes the LPS-binding region (residues 28–34),20 but when isolated this peptide fragment folds into a ß-hairpin structure.21 Peptides corresponding to this region exhibit antibacterial activity against Gram-positive and Gram-negative bacteria19 and bind LPS.19,22

Polymyxin B is one of the most potent neutralizers of LPS. Removal of the 6-heptanoyl/octanoyl diaminobutyryl moiety results in loss of antibacterial activity.23 This led us to explore the influence of lipophilic modification of a peptide based on residues 21–31 of human lactoferrin (LF12) on antibacterial activity and on LPS-binding and neutralizing activity. The method we used for obtaining recombinant antibacterial peptide allows straightforward preparation of lipopeptide conjugates24 and potentially cost-effective large-scale production. By modification with acyl chains, we have enhanced its antibacterial activity against Gram-negative, and to a greater extent, Gram-positive bacteria. Endotoxin binding and in vitro neutralization were enhanced >10-fold.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents

The chemicals used were of the highest quality commercially available and obtained mostly from the Sigma-Aldrich Corporation (St Louis, MO, USA). A stock solution of lipid A (hexa-acyl lipid A from F515 Escherichia coli, provided by Dr Ulrich Zaehringer), was prepared (1 mg/mL) in endotoxin-free water, sonicated for 10 min and stored in small aliquots at –20°C. On the day of use it was thawed and sonicated for 3 min. Laboratory glassware used in the chromogenic Limulus amoebocyte lysate (LAL) assay was thoroughly cleaned and baked dry for 4 h at 180°C to render it free of contaminating LPS. Pipette tips from their original packing were wrapped in aluminium foil piece by piece and autoclaved for 45 min at 131°C and 1.2 x 105 Pa.

Bacterial strains and growth conditions

E. coli DC2 (CGSC 7139) was obtained from the E. coli Genetic Stock Centre (Yale University, New Haven, CT, USA). Staphylococcus aureus (ATCC 25923) was obtained from the American Type Culture Collection (Manassas, VA, USA). Bacterial cultures were stored at –70°C and grown on Luria–Bertani (LB) medium at 37°C.

Preparation and purification of lipopeptides

Procedures for cloning, production and purification of the recombinant dodecapeptide LF12 (FQWQRNIRKVR-homoserine lactone) were as described for the production of 15N-enriched peptide [15N]LF12.24 Peptide LF12 was derived as follows: 170–460 nmol of purified recombinant LF12 was dried in a centrifuge evaporator, completely lactonized by the addition of 100% trifluoroacetic acid (TFA) 20 µL and dried in a rotary evaporator. The dried pellet was dissolved in anhydrous N, N-dimethylformamide (DMF) 50 µL, delivered by a gas-tight syringe, and triethylamine (Et3N) 8 µL was added. Alkylamine (hexylamine, n-octylamine, dodecylamine, tetradecylamine, hexadecylamine, oleylamine) at 100-fold molar excess over peptide was added to the lactonized peptide solution and incubated overnight at 45°C. Lipopeptides were isolated by reverse-phase (RP)-HPLC and eluted at room temperature with a linear gradient from 30% to 70% of buffer B (80% acetonitrile, 0.05% TFA in deionized and degassed water) over 20 min and from 70% to 100% of buffer B over 10 min, at a flow rate of 0.5 mL/min. The identity of the lipopeptides was confirmed by fast atom bombardment mass spectrometry (FAB-MS) using a mass spectrometer, AutoSpec (MicroMass, Manchester, UK).

Spectroscopic characterization of lipopeptides and their interaction with lipid A

A PTI (Photon Technology International, Lawrenceville, NJ, USA) spectrofluorimeter was used to measure the intrinsic tryptophan fluorescence of the lipopeptides. Emission spectra were recorded from 320 to 370 nm at 25°C in a 10 mm quartz cuvette with excitation at 280 nm. Slit widths were set at 1 nm. Fluorescence measurements were similarly used to characterize peptide binding to lipid A. Binding of lipopeptides to lipid A in 20 mM K-phosphate buffer pH 7.0 was monitored by observing the change in the intrinsic tryptophan fluorescence of each lipopeptide. Lipid A was gradually added to a fixed amount of peptide (1 µM) to a final concentration from 0.5 to 6 µM. From the fit of the fluorescence versus lipid A concentration curve to the equation: F = Fmax {Kd + P0 + L0{surd}[(Kd P0 – L0)2 – 4P0L0]}/2 where F is fluorescence intensity, Fmax maximal fluorescence intensity, Kd dissociation constant, P0 total peptide concentration and L0 total lipid A concentration, taking into account ligand (lipid A) depletion,25 the dissociation constant was determined for each peptide using a non-linear curve fit implemented in the program Origin (Microcal).

In vitro LPS binding

The ability of lipopeptides to neutralize LPS in vitro was assayed by the chromogenic LAL test26 according to the manufacturer’s instructions (Cape Cod Associates, Falmouth, MA, USA). LPS [3.2 endotoxin units (EU)/mL] was mixed with various concentrations of peptides in endotoxin-free water. A total of 50 µL of each mixture was added to an equal volume of the pyrochrome reagent in endotoxin-free water and incubated for 22 min at 37°C in a 96-well endotoxin-free microtitre plate pre-equilibrated at 37°C. The reaction was terminated by adding acetic acid to 10%. The absorbance at 405 nm was read with a microplate reader 3550-UV (Bio-Rad, Hercules, CA, USA).

Determination of antibacterial activity

Two methods were used to assay the antibacterial activity of the lipopeptides. In an agar plate assay,27 overnight cultures of E. coli and S. aureus in LB medium (tryptone 10 g/L and yeast extract 5 g/L) were diluted 1:10 into a top agar (6% bacteriological agar in LB medium). Two millilitres of the top agar lawned with bacteria was poured over LB agar plates warmed at 37°C. Ten microlitres of different concentrations of peptides were spotted on to solidified top agar. Peptides were serially diluted in sterile deionized water in a final volume of 10 µL. The plates were incubated at 37°C for 3 h.

Additionally, the antibacterial activity of lipopeptides was assayed on E. coli and S. aureus using a cfu assay:28 cells were incubated with various concentrations of peptides at 37°C for 4 h in 10 mM sodium phosphate buffer (pH 7.4), or at 37°C for 2 h in LB medium, and the number of cfu was determined by plating the diluted cell suspension on to LB agar plates.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation and purification of LF12 lipopeptides

Production of recombinant peptides in bacteria may be a cost-efficient alternative to chemical synthesis, particularly for isotope-labelled peptides. The bacterial production of peptides requires a special approach because of their sensitivity to proteolytic degradation. An additional problem is the toxicity of the peptides to bacteria that produce them. Both problems were solved by producing the peptides in the form of insoluble fusion proteins.29 We have used peptide fusion with ketosteroid isomerase (KSI), a protein that is highly insoluble in water as well as in the cytoplasm of bacteria.24 We prepared peptide LF12, with Met-27 in the original sequence of human lactoferrin replaced by isoleucine, which is found at position 27 in the porcine variant of lactoferrin.19 This modification was necessary because of the use of cyanogen bromide (CNBr) for cleavage of the fusion protein between the peptide and carrier protein. Additionally, potential oxidation of the methionine residue was avoided. This modification did not change the antibacterial activity of the peptide.19 Yields of inclusion bodies of KSI–LF12–His6 fusion protein30 exceeded 400 mg/L of the bacterial culture in LB medium. Cleavage of KSI–LF12–His6 fusion protein with CNBr released dodecapeptide LF12, containing, owing to the CNBr cleavage, a reactive homoserine-lactone group at its C-terminus. KSI ‘peptide carrier protein’, which is hydrophobic, precipitated, while the LF12 peptide was present in the solution and was separated from other peptides (i.e. terminal His6-containing C-terminal peptide) by RP-HPLC. A range of LF12 conjugates was prepared by reaction with alkylamines with substituent hydrocarbon chains ranging from six to 18 carbon units. Reaction products were separated by RP-HPLC and their identity confirmed by mass spectra (Table Go). The maximum in emission fluorescence wavelength ({lambda}max) decreased with increasing acyl chain length up to 12 carbon units (LF12-C12) and then increased again for longer chain lengths (data not shown). This may be because the addition of acyl chains up to 12 carbon units increased the hydrophobicity of the environment of tryptophan, whereas the lipopeptides with longer acyl chains may have formed micelles or other aggregates with aliphatic domains segregated from the tryptophan residue, as recently suggested for lipophilic acid-modified magainin.31


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Table 1.  Properties of synthesized (lipo)peptides
 
Lipid A binding and in vitro neutralization of LPS

Dissociation constants of lipopeptides to lipid A were determined by fluorescence titration. Binding of lipid A to each peptide resulted in a blue-shift and an intensity increase of the fluorescence emission spectra. From the fit of the fluorescence versus concentration curves, the dissociation constant for each peptide was determined (Figure 1, Table Go). Derivatization of LF12 with the C12 chain (LF12-C12) enhanced its binding to lipid A 14-fold. Its Kd at 1.5 µM was only three-fold higher in comparison with polymyxin B.32 In vitro, the LPS neutralization potency of lipopeptides was determined using the LAL assay. The most potent inhibitor of the LAL reaction was again LF12-C12, where the neutralizing concentration at 2.4 µM was only two-fold higher in comparison with polymyxin B (Table Go)33 and was 12-fold lower than the 50% endotoxin-neutralizing concentration (ENC50) of the parent peptide LF12.



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Figure 1. Binding of LF11-C12 and LF12 peptides to lipid A as monitored by intrinsic tryptophan fluorescence. Upper curve: LF12-C12; lower curve: LF12. Lipid A was added to 1 µM solution of peptide in 20 mM K-phosphate buffer pH 7.0. Solid lines represent the best non-linear fits as described in the Materials and methods section. Fraction of the complex was determined from (FF0)/Fmax (F0, fluorescence intensity at 330 nm without ligand; Fmax, fluorescence intensity at ligand saturation). Only a low ligand concentration range is shown for the LF12 experiment (measured to 50 µM).

 

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Table 2.  Antibacterial and endotoxin-binding activities of (lipo)peptides
 
Antibacterial activity

A cfu assay was used for determination of MICs. The antibacterial activities of peptides were also determined by agar plate assay. MIC values in the cfu assay were lower by up to two orders of magnitude in the solution assay, in comparison with the agar plate assay, which is probably due to the interactions of peptides with agar or high inocula of bacteria. All lipopeptides were more effective than the parent peptide LF12 against both Gram-negative and Gram-positive bacteria (Table Go) and showed higher antibacterial activity against E. coli than against S. aureus (Figure 2). The optimal acyl chain length was 12 carbon units, with the highest enhancement of antibacterial activity of 50-fold against E. coli, and 78- or 75-fold enhancement of activity against S. aureus when compared with the parent peptide. The results of the assay in sodium phosphate buffer showed similar antibacterial activity of peptides against both types of bacteria (Table Go, Figure 2).



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Figure 2. Enhancement of antibacterial activity by modification of LF12 peptides with acyl chains. Ratios of MIC were calculated for E. coli and S. aureus as [MIC(LF12)]/[MIC(lipopeptide)] in buffer (a) and in LB medium (b).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we have improved the antibacterial activity of a peptide based on a human protein by the addition of acyl chain. Polymyxin B nonapeptide, a derivative of polymyxin B lacking the 6-heptanoyl/octanoyl diaminobutyryl group of the parent compound, has no antibacterial activity and poor anti-endotoxic activity.23 NMR experiments have shown that the interaction between polymyxin B and LPS involves electrostatic interactions between the polar head group of lipid A and the charged residues of polymyxin B, and hydrophobic interactions between the lipid chains of LPS and the acyl chain, as well as of a cluster of hydrophobic residues on polymyxin B.34

We have selected LF12, a peptide based on human lactoferricin, as a host peptide in the design of novel antibacterial compounds. MIC values of peptides based on human, mouse and goat lactoferricins are in the range 63–240 µM for E. coli and S. aureus, whereas the bovine variant is more potent at 9 µM.35 This higher antibacterial activity is believed to be the result of the additional tryptophan residue. The added hydrophobic chain in our lipopeptides probably fulfils the same function as the tryptophan residue, which was found to penetrate into the membrane as a hydrophobic anchor.36

The dissociation constants of lipopeptides for their binding to lipid A were similar to the neutralizing concentrations in LAL, confirming that lipopeptides bound specifically to the lipid A moiety. Our results suggest that LF12 lipopeptides bind to LPS in a similar manner to polymyxin B:34,3739 both electrostatic interactions, particularly between cationic residues of the lipopeptide and phosphate groups of LPS, and hydrophobic interactions between the acyl chain of the lipopeptide and the lipid chains of LPS contribute to the interaction in solution. The acyl moiety of the lipopeptide is probably important for disorganizing the bacterial membrane by disrupting the lipid packing and the supramolecular structure of LPS, which is important for its endotoxic activity.

Results on LF12 lipopeptides are similar to the data on modification of peptide antibiotic octapeptin, where the best antibacterial activity was achieved at chain lengths of C8 for E. coli and C12 and C14 for Bacillus subtilis.40 We have observed that the distribution of antimicrobial activity as a function of acyl chain length was skewed towards longer chains for Gram-positive bacteria. Improvement of antibacterial activity by peptide acylation was higher for S. aureus than for E. coli. This indicates that antibacterial specificity as well as efficiency can be altered by the nature of the hydrophobic substituent.

The efficiency of (lipo)peptides depended on the composition of the medium with complex medium (LB) increasing the MIC by up to15-fold, in comparison with the buffer at low ionic strength (Table Go). The presence of sodium chloride (171 mM) in the medium did not affect the antibacterial activity against Gram-negative and Gram-positive bacteria (data not shown). This is important for their potential therapeutic use, because many antibacterial peptides have reduced activity under physiological or increased salt conditions (e.g. chronic inflammation of lungs by patients with cystic fibrosis).41,42

The modification of antibacterial peptide with acyl chains has the potential for further improvement. Human peptides and fragments of proteins are an attractive source of host peptides, since they are less likely to cause antigenic reaction. Human proteins that interact with LPS more effectively than lactoferrin (e.g. LPS-binding protein, bactericidal/permeability-increasing protein),4345 or which are involved in activation of immune cells caused by LPS (CD14, TLR4, MD-2),4648 are particularly interesting as future donors of host peptides.


    Acknowledgements
 
We thank Dr Ulrich Zaehringer from the Forschungscentrum, Borstel, Germany for lipid A, Robert Bremak for his excellent technical help, Professor Rober H. Pain for his comments on the manuscript, and Bogdan Kralj and Du&[scaron];an &[Zcaron];igon (The National Mass Spectrometry Center at the Jo&[zcaron];ef Stefan Institute in Ljubljana, Slovenia) for measuring the mass spectra of lipopeptides. This research was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia.


    Footnotes
 
* Corresponding author. Tel: +386-1-476-0372; Fax: +386-1-476-0300; E-mail: roman.jerala{at}ki.si Back


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 Materials and methods
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 Discussion
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