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

Abstract

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.

Full text

Journal of Antimicrobial Chemotherapy (2003) 51, 1159–1165
DOI: 10.1093/jac/dkg219
Advance Access publication 14 April 2003
1159
...................................................................................................................................................................................................................................................................
Published by Oxford University Press
Enhancement of antibacterial and lipopolysaccharide binding activities
of a human lactoferrin peptide fragment by the addition of acyl chain
Andreja Majerle, Jurka KidriQ 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
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
Animals and plants produce cationic antibacterial peptides as
a first line of defence against invading pathogenic micro-
organisms.
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 phospho-
lipids. Development of resistance against such peptides, by
modification of the membrane composition, is unlikely with-
out compromising the bacterial viability. Antibacterial pep-
tides 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 bac-
teria, 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 cyto-
kines, adhesion molecules and others are produced
7
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 pep-
tides 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 neutro-
phils during inflammatory responses.
12
It has antibacterial
activity against a broad range of Gram-positive and Gram-
negative bacteria and fungi.
13–15
Human lactoferrin binds to
lipid A with high affinity
16
and induces LPS release from the
cell wall of Gram-negative bacteria.
17
Proteolytic digestion of
human lactoferrin yields a peptide fragment called lacto-
ferricin H, which has enhanced antibacterial activity compared
to intact lactoferrin.
18
Lactoferricin contains a region that
forms an amphipathic α-helix (residues 21–31), distinct from
the site of iron binding
19
and includes the LPS-binding region
(residues 28–34),
20
but when isolated this peptide fragment
folds into a β-hairpin structure.
21
Peptides corresponding to
this region exhibit antibacterial activity against Gram-
positive and Gram-negative bacteria
19
and bind LPS.
19,22
Polymyxin B is one of the most potent neutralizers of LPS.
Removal of the 6-heptanoyl/octanoyl diaminobutyryl moiety
..................................................................................................................................................................................................................................................................
*Corresponding author. Tel: +386-1-476-0372; Fax: +386-1-476-0300; E-mail: roman.jerala@ki.si
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A. Majerle et al.
1160
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
conjugates
24
and potentially cost-effective large-scale pro-
duction. 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
Reagents
The chemicals used were of the highest quality commercially
available and obtained mostly from the Sigma-Aldrich Cor-
poration (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 × 10
5
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
15
N-enriched peptide [
15
N]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 (Et
3
N) 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 character-
ize 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 fluores-
cence 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 = F
max
{K
d
+ P
0
+ L
0
–
√[(K
d
– P
0
– L
0
)
2
– 4
P
0
L
0
]}/2 where F is fluorescence
intensity, F
max
maximal fluorescence intensity, K
d
dissocia-
tion constant, P
0
total peptide concentration and L
0
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 test
26
according to the
manufacturer’s instructions (Cape Cod Associates, Fal-
mouth, 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
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Addition of acyl chain to enhance antibacterial peptides
1161
extract 5 g/L) were diluted 1:10 into a top agar (6% bacterio-
logical 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 pep-
tides 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
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 pep-
tides 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 prod ucing the peptides in the form of insoluble
fusion proteins.
29
We have used peptide fusion with keto-
steroid 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–His
6
fusion protein
30
exceeded 400 mg/L of the bacterial culture in LB medium.
Cleavage of KSI–LF12–His
6
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 hydro-
phobic, precipitated, while the LF12 peptide was present in
the solution and was separated from other peptides (i.e. ter-
minal His
6
-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 separ-
ated by RP-HPLC and their identity confirmed by mass
spectra (Table 1). The maximum in emission fluorescence
wavelength (λ
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 trypto-
phan, 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
Lipid A binding and in vitro neutralization of LPS
Dissociation constants of lipopeptides to lipid A were deter-
mined 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 fluores-
cence versus concentration curves, the dissociation constant
for each peptide was determined (Figure 1, Table 2). Derivatiz-
ation of LF12 with the C
12
chain (LF12-C12) enhanced its
binding to li pid A 14-fold. Its K
d
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 1)
33
and was 12-fold lower than the 50%
endotoxin-neutralizing concentration (ENC
50
) of the parent
peptide LF12.
Antibacterial activity
A cfu assay was used for determination of MICs. The anti-
bacterial 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 inter-
actions 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 2) and showed higher antibacterial activity against
E. coli than against S. aureus (Figure 2). The optimal acyl
Tabl e 1 . Properties of synthesized (lipo)peptides
a
Isolation by RP-HPLC (linear gradient from 30% to 70% of buffer B over
20 min and from 70% to 100% of buffer B over 10 min, at a flow rate of
0.5 mL/min and room temperature).
b
Determined by FAB-MS.
Peptide
Elution at %
of buffer B
a
Molecular mass experimental
b
(calculated) (Da)
LF12 45 1613.6 (1613.8)
LF12-C6 52 1715.0 (1714.9)
LF12-C8 54 1743.2 (1743.2)
LF12-C12 58 1798.7 (1798.7)
LF12-C14 72 1828.1 (1827.2)
LF12-C16 76 1854.3 (1854.3)
LF12-C18 80 1881.6 (1881.3)
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A. Majerle et al.
1162
chain length was 12 carbon units, with the highest enhance-
ment 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 2,
Figure 2).
Discussion
In the present study, we have improved the antibacterial activ-
ity of a peptide based on a human protein by the addition of
acyl chain. Polymyxin B nonapeptide, a derivative of poly-
myxin 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 lacto-
ferricin, 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:
34,37–39
both electrostatic interactions, particularly between cationic
residues of the lipopeptide and phosphate groups of LPS, and
Figure 1. Binding of LF11-C12 and LF12 peptides to lipid A as moni-
tored 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 th e best non-linea
r
fits as described in the Materials and methods section. Fraction of the
complex was determined from (F – F
0
)/F
max
(F
0
, fluorescence intensity
at 330 nm without ligand; F
max
, fluorescence intensity at ligand satura-
tion). Only a low ligand concentration range is shown for the LF12
experiment (measured to 50 µM).
Tabl e 2 . Antibacterial and endotoxin-binding activities of (lipo)peptides
K
d
, dissociation constant; ND, not determined.
a
ENC
50
of peptide in the chromogenic LAL test using 3.2 EU/mL of LPS. ENC
50
values (means ± S.E.M.) were
calculated by sigmoidal curve fitting.
b
Cfu assay, incubation in 10 mM sodium phosphate buffer (pH 7.4).
c
Cfu assay, incubation in LB medium.
Lipid A-binding
activity
In vitro LPS
neutralization Antibacterial activity (µM)
Peptide K
d
(µM) ENC
50
(µM)
a
E. coli
b
E. coli
c
S. aureus
b
S. aureus
c
LF12 20.7 30.0 ± 2.4 40 200 200 >600
LF12-C6 6.41 11.2 ± 2.2 1.5 25 15 25
LF12-C8 3.21 8.84 ± 3.3 2 19 10 19
LF12-C12 1.50 2.43 ± 1.6 0.3 4 8 8
LF12-C14 3.35 19.7 ± 1.0 0.6 7 15 15
LF12-C16 7.93 12.1 ± 1.4 3 7 14 14
LF12-C18 8.95 ND 5 8 ND ND
Polymyxin B 0.50
32
1.4 ± 0.5 0.2 0.2 2 2
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Addition of acyl chain to enhance antibacterial peptides
1163
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 struc-
ture 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
C
8
for E. coli and C
12
and C
14
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 com-
position of the medium with complex medium (LB) increas-
ing the MIC by up to15-fold, in comparison with the buffer at
low ionic strength (Table 2). The presence of sodium chloride
(171 mM) in the medium did not affect the antibacterial activ-
ity 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),
43–45
or which are involved in activation of
immune cells caused by LPS (CD14, TLR4, MD-2),
46–48
are
particularly interesting as future donors of host peptides.
Acknowledgements
We thank Dr Ulrich Zaehringer from the Forschungscentrum,
Borstel, Germany for lipid A, Robert Bremšak for his excel-
lent technical help, Professor Rober H. Pain for his comments
on the manuscript, and Bogdan Kralj and Dušan higon (The
National Mass Spectrometry Center at the Joief Stefan Insti-
tute 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.
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