Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial α-helix of human lactoferrin

Article abstract of DOI:10.1128/AAC.00908-09

There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial α-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial α-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential. Copyright

Full text of DOI:10.1128/AAC.00908-09

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2010, p. 418–425
0066-4804/10/$12.00 doi:10.1128/AAC.00908-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 1
Structure-Microbicidal Activity Relationship of Synthetic Fragments
Derived from the Antibacterial ␣-Helix of Human Lactoferrinᰔ
L. Håversen,1,2 N. Kondori,1 L. Baltzer,3† L. Å. Hanson,2 G. T. Dolphin,3
K. Dun´er,1 and I. Mattsby-Baltzer1*
Departments of Infectious Medicine/Clinical Bacteriology1 and Clinical Immunology,2 University of Gothenburg, Gothenburg,
Sweden, and Department of Chemistry, Linko¨ping University, Linko¨ping, Sweden3
Received 2 July 2009/Returned for modification 21 August 2009/Accepted 29 October 2009
There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria
and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31
(sequence 14-31) from the N-terminal end, corresponding to the antibacterial ␣-helix of human lactoferrin
(LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis
(99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida
albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14–31 showed
that the peptide sequence 19–31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial
activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in
a Limulus assay. Alanine scanning of peptide sequence 20–31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31
was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral
hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal
activity significantly against all test organisms compared to the amino acids natural counterpart, also, in
comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was
less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as
tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences,
based on the antimicrobial ␣-helix region of LF, can be significantly enhanced by optimizing the length and
substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.
Steadily increasing antimicrobial drug resistance has become
a worldwide problem. For instance, resistance to multiple an-
tibiotics can be found among pathogens such as staphylococci,
pneumococci, Pseudomonas species, and extended-spectrum
␤-lactamase (ESBL)-producing strains of Enterobacteriaceae.
Also, resistance to antimycotics, such as the azoles, has climbed
significantly. Thus, there is a need for new treatment therapies.
Human lactoferrin (LF), a single-chain iron-binding glyco-
protein, is a major protein in milk and mucosal secretions. It is
also found in the secondary granulae of neutrophils. It is a
multifunctional molecule participating in the innate host de-
fense, expressing both antimicrobial and antiinflammatory ac-
tivities in vivo (16–18, 34).
Iron depletion and destabilization of the bacterial cell
wall are two mechanisms suggested to be involved in the
antimicrobial activity of lactoferrin. In Gram-negative bac-
teria, destabilization could partly be due to its binding ca-
pacity to lipid A and porins (3, 4, 10, 25, 37). Binding to
lipopolysaccharide (LPS) has been suggested to release LPS
from the bacterial membrane. A third mechanism of its
antimicrobial activity is the release of microbicidal frag-
ments upon enzymatic hydrolysis of LF (4).
The pepsin-derived bactericidal LF fragment, lactoferricin
(Lfcin), consists of residues 1–47 or 49 from the N-terminal
end, comprising two antimicrobial domains (residues 1–11 and
18–40) (4, 19, 26). Another recently discovered antimicrobial
region of the LF molecule comprises residues 153–183 (32). A
smaller peptide sequence of LFcin (residues 21–31), corre-
sponding to a part of the first ␣-helix of the LF molecule, has
been studied in more detail (5). This 11-amino-acid-long pep-
tide exhibited bactericidal activity against Escherichia coli and
caused disruption of the outer membrane of E. coli O111 (6,
27). The amphipathic ␣-helix region also comprises a part of
the LPS and glycosaminoglycan binding region of LF, as de-
termined by site-directed mutagenesis at positions 28–34 in the
LF molecule (9, 23). The amino acid residues 35–40 constitute
a ␤-sheet region in the native LF molecule (2, 15). Although
LFcin may not have a well-developed secondary structure in
solution, it may adopt a helical structure when bound to mem-
branes, analogous to other antimicrobial peptides (15).
In the present study, the aim was to investigate the structure-
microbicidal activity relationship of peptide fragments based on the
antibacterial ␣-helix (amino acid residues 14–31) of LF, using E. coli,
Staphylococcus aureus, and Candida albicans as test organisms. The
experimental strategy included downsizing the helical region, fol-
lowed by alanine scanning and amino acid substitutions.
* Corresponding author. Mailing address: Department of Infectious
Medicine/Clinical Bacteriology, University of Gothenburg, Guldheds-
gatan 10, 413 46 Gothenburg, Sweden. Phone: 46-(0)31 3424728. Fax:
46-(0)31 3424975. E-mail: inger.mattsby-baltzer@microbio.gu.se.
† Present address: Department of Biochemistry and Organic Chem-
istry, Uppsala University, Uppsala, Sweden.
MATERIALS AND METHODS
Bacterial and fungal strains. The following bacterial strains and yeast strain
were used in the microplate assay: Klebsiella pneumoniae (CCUG 9997), Entero-
coccus faecalis (ATCC 19433), Staphylococcus epidermidis (CCUG 18000A),
Pseudomonas aeruginosa (CCUG 551), Staphylococcus aureus 1800 (CCUG
ᰔ Published ahead of print on 16 November 2009.
418

VOL. 54, 2010
MICROBICIDAL ACTIVITY OF LACTOFERRIN-DERIVED PEPTIDES
419
TABLE 1. Microbicidal activities of peptide sequencesa
TABLE 2. Antimicrobial activities of peptide sequencesa
99% killing activityb
MMC99 ␮g/ml
Peptide
sequence
E. coli
S. aureus
2 h 20 h
C. albicans
P valueb
2 hc
20 h
2 h
20 h
Peptide
sequence
P value
18–31
19–31 (HL9)
20–31
50
25
100
25
12
50
50
25
100 Ͼ200
50
50
50
200
Ͻ0.05b
Ͼ100 Ͼ100 Ͼ200 100
a Shown are antimicrobial activities of peptide sequences containing amino
acid residues 18-31 (TKCFQWQRNMRKVR), 19-31 (KCFQWQRNMRKVR),
and 20-31 (CFQWQRNMRKVR) using twofold serial dilutions, starting with
100 or 200 ␮g/ml in BP, and incubated for 2 and 20 h with E. coli O6K5, S. aureus
1800, or C. albicans. All samples were run in duplicate.
b Values were determined using the Friedman test with Dunn’s multiple com-
parison test.
14–31
15–31
16–31
17–31
18–31
19–31
20–31
21–31
22–31
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
0.0026c
}
}
c h, hours of incubation.
Ͻ0.0001
a Shown are microbicidal activities of peptide sequence 14-31 (QPEATKCF
QWQRNMRKVR) and of the series of peptides resulting from the removal of
one amino acid at a time from the N-terminal end of peptide sequence 14-31 to
the sequence 22-31. Microbial strains were incubated in BHIdil for 2 h at 37°C in
the presence of 25 ␮g/ml of each peptide, except for E. coli O6 and C. albicans,
which were incubated with 10 ␮g/ml. All samples were run in duplicate.
b ϩ, at least 99% killed by 25 ␮g/ml or 10 ␮g/ml for E. coli O6 and C. albicans;
Ϫ, Ͻ99 % killed.
mixture was left with stirring and air contact for approximately 1 day and then
lyophilized.
All peptides were purified by reverse-phase high-performance liquid chroma-
tography (HPLC) and eluted with isochratic mixtures of isopropanol and 0.1%
TFA. After lyophilization, the peptides were dissolved in distilled water and the
purities of the peptides were determined, by HPLC, to be Ն95%. The masses of
the peptides were analyzed by electrospray mass spectrometry, using a VG Zab
spectrometer sector instrument, and the experimentally determined molecular
masses were within 1 mass unit of the theoretical ones for the peptides.
MMC99. Washed cells were suspended in diluted BHI medium (1/100 of 3.4%,
pH 6.7 to 6.9) (BHIdil) or suspended in 1% Bactopeptone (BP) (pH 7.0) (Difco).
The absorbance of the suspension was measured by spectrophotometry at 620
nm and diluted to a concentration corresponding to 4 ϫ 106 cells/ml. Peptides
serially diluted in BHIdil or BP by twofold steps (unless otherwise stated) were
added to the wells of a microtiter plate in duplicate or triplicate (200 ␮l per well).
The bacterial or yeast cell suspensions were added in 10-␮l volumes to give a final
concentration of approximately 2 ϫ 105 cells/ml. The concentration of the inoc-
ulum suspension was always checked by viable counts. The microplate was
incubated at 37°C in a humid chamber for 2 h unless stated otherwise. Five
microliters was taken from each well and added as a drop onto a blood agar plate
and incubated, overnight, at 37°C. The viable count in each area as formed by the
drop was recorded, and the concentration of CFU/ml was calculated. A killing
activity of between 95 and 99.9% (corresponding to 1 to 50 CFU per drop area)
could be calculated. Thus, no growth corresponded to less than 200 CFU/ml,
indicating a killing of more than 99.9%. The minimum concentration of a peptide
c Values were determined using the chi-square test for trends.
1800), methicillin-resistant Staphylococcus aureus (MRS3526), E. coli O6K5 and
O14, and Candida albicans (ATCC 64549). All microorganisms were cultured in
brain heart infusion medium (BHI) on a shaker overnight at 37°C. A volume of
the culture was transferred to a new tube with BHI and incubated for two
more hours on the shaker. The microorganisms were harvested at the log
phase of growth, washed once, and suspended in the broth used for the
microplate assay.
LF. Lactoferrin from human milk was purchased from Sigma (Saint Louis,
MO). The level of iron saturation was approximately 7%.
Peptide synthesis: Fmoc solid-phase peptide synthesis of human lactoferrin
bactericidal domain (HLBD) mimics. Peptides were synthesized using a contin-
uous-flow Applied Biosystems Pioneer automated peptide synthesizer and stan-
dard 9H-fluoren-9-ylmethoxy carbonyl (Fmoc) protection group protocols. The
peptides were synthesized on
a 0.1- to 0.2-mmol scale, using the resins
4-hydroxymethylphenoxy–acetic acid polyethylene glycol–polystyrene (PAC-
PEG-PS; Applied Biosystems) (0.21 mmol/g) for the peptide acids and Fmoc–5-
(4-aminophylline-3, 5-dimethoxyphenoxy) valeric acid–PEG-PS (Fmoc-PAL-PEG-
PS) (0.20 mmol/g) for the peptide amides.
Side chains were protected with the piperidine-stable tert-butyl (Ser and Thr),
tert-butyl ester (Glu), tert-butyloxycarbonyl (Lys and Trp), triphenylmethyl (Asn,
Cys, Gln, and His), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
(Arg) groups.
The Fmoc ␣-amino protecting group was removed by treatment with 20%
piperidine in dimethylformamide (DMF) for 7 min. All couplings proceeded in
DMF, with a four-times excess of activated amino acid over the growing peptide
chain, using 4 eq of O-benzotriazole-1-yl-1,1,3,3-tetramethyluroniumtetraflu-
oroborate/diisopropylethylamine (1:2, mol/mol) and 4 eq of amino acid. A
sixfold excess of hydroxybenzotriazole was added in the coupling of the
cysteine residue.
causing Ն99% reduction of the inoculum was defined as MMC99
.
In one experiment analyzing the kinetics of killing (see Fig. 2), serial dilutions
of the samples of the microplate assay were performed and cultured.
Limulus assay. Peptides were analyzed for their capacity to neutralize LPS
(O111:B4, O55:B5; Sigma) and lipid A (monophosphoryl and diphosphoryl,
L6895, L0774; Sigma) by the Limulus assay (LAL activity) (coamatic endotoxin,
chromogenic endpoint; Chromogenix, Sweden). Pyrogen-free Tris buffer (0.05
M, pH 7.3) was used as diluent. Also, two LPS-neutralizing agents, polymyxin B
(Sigma) and a peptide sequence of the bactericidal/permeabilization-increasing
protein (BPI peptide) (KISGKWAQKRFLKMSGNF; MedProbe), were in-
cluded in the analysis. The peptides (200 ␮l) were serially diluted in two- and
fivefold steps, and each dilution was added to glass tubes containing LPS/lipid A
in Tris buffer (200 ␮l). The concentration of LPS/lipid A monophosphoryl was
0.3 ng/ml, and that of lipid A diphosphoryl was 0.15 ng/ml (approximately 0.300
to 1.0 EU/ml). The tubes were incubated for 1 h at 37°C. The remaining Limulus
activity after the incubation was analyzed according to instructions in the man-
ufacturer’s manual.
Peptides were capped by acylation of the amino terminal by treatment with a
0.3 M solution of acetic anhydride in DMF for 10 min.
Final deprotection and cleavage of the peptide from the resin was achieved by
treatment with a mixture of 9.25 ml trifluoroacetic acid (TFA), 250 ␮l water, 250
␮l ethanedithiol, and 250 ␮l triisopropylsilane per gram of peptide resin for 2 h
at room temperature. The resin was removed by filtration, and the peptide was
precipitated twice by cold diethyl ether, centrifuged, and resuspended in fresh
diethyl ether to extract the scavengers and TFA. The peptides were dissolved in
water and lyophilized.
Statistics. Chi-square analyses for trends were used for comparing a series of
peptides with decreasing/increasing lengths of the peptide sequence (Table 1).
The Friedman test with Dunn’s multiple comparison test was used for compar-
ison of matched samples of several peptides against one specific peptide (Tables
2 and 3).
In the microbicidal microplate assay, peptide solutions serially diluted twofold
and differing by twofold titer steps (a fourfold difference in MMC99) were
significantly different.
A cyclic disulfide-bridged peptide was prepared from crude, unpurified mate-
rial. Approximately 200 mg of peptide was dissolved in 1 liter of deaerated water.
Ammonium hydrogen carbonate was added to adjust the pH to 6 to 7, and the

420
HÅVERSEN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Microbicidal activities of peptidesa
The importance of single amino acids for microbicidal ac-
tivity. (i) Alanine scanning. One amino acid was replaced, at
each position, in the peptide sequence 20–31 by Ala to deter-
mine the effect of each individual amino acid for antimicrobial
activity (Table 3). The peptides were analyzed against E. coli,
S. aureus, and C. albicans.
MMC99 ␮g/ml
Residue
Amino
acid
position
replaced
by
BHIdil
BP
P
valueb
E.
S.
C.
E.
coli
S.
C.
alanine
coli aureus albicans
aureus albicans
A significant reducing effect on the killing activity against E.
coli, using BHIdil, was obtained with an Ala substitution at
position 20, where a Cys residue was replaced (Table 3).
In BP, a significantly reduced antimicrobial activity against
C. albicans was seen, not only for the Cys replacement, but also
for the Trp replacement at position 23, the replacement of Arg
residues at positions 28–31 and the Lys replacement at position
29. A similar pattern was observed for E. coli, although not
significantly different compared to the original peptide.
Changing the hydrophilic Gln (position 22 or 24) or Asn
(position 26) to a slightly hydrophobic Ala resulted in signifi-
cantly increased overall antimicrobial activity of the peptide
with Ala at position 26 (P Ͻ 0.05; the Friedman test with
Dunn’s multiple posttest). MMC99 values for the peptide with
Ala at position 22 were reduced compared to those of the control
peptide for all microorganisms and media, although not signifi-
cantly.
A dramatic difference in the concentration of peptide
needed for 99% killing between the two growth media was
seen for S. aureus (up to 30 times or more), while the difference
was less for E. coli and C. albicans (four- to eightfold).
(ii) Substitution by hydrophobic or charged amino acids. In
all the following amino acid replacement experiments, the mi-
crobicidal assays were run in both BHIdil and BP. There was,
however, no significant difference in antimicrobial activity be-
tween the natural peptide sequence 20–31 and the peptides
with the substitutions listed in Table 4 for experiments per-
formed in BHIdil, except for one peptide. Thus, all results
described below refer to assays performed in BP.
20
21
22
23
24
25
26
27
28
29
30
31
C
Ͼ25 14
12
12
6
12
12
12
6
25
25
25
25
25
12
Ͼ100 Ͼ100 Ͼ100
ND ND ND
50 100 25
Ͼ100 Ͼ100 Ͼ100
50 100 50
ND ND ND
25 50
50 Ͼ100
F
12
6
7
Q
W
Q
R
N
M
R
K
V
R
3.5
25 14
12
12
6
12
25
12
12
12
12
3.5
7
3.5
3.5
7
25 Ͻ0.05
50
Ͼ100 Ͼ100 Ͼ100
100 Ͼ100 Ͼ100
3.5
3.5
7
50 Ͼ100
Ͼ100 Ͼ100 Ͼ100
100 Ͼ100 50
50
None None
7
a Shown are microbicidal activities of peptides derived from amino acid resi-
dues 20-31 (CFQWQRNMRKVR) by alanine scan. Peptides were incubated
with C. albicans, E. coli O6K5, and methicillin-resistant S. aureus MRS3525 in
BHIdil or BP. The peptide concentrations were serially diluted in twofold steps,
starting with 25 or 28 ␮g/ml (S. aureus) using BHIdil and 100 ␮g/ml using BP as
growth medium. All samples were run in duplicate. ND, not determined.
b Values were determined using the Friedman test with Dunn’s multiple com-
parison test. Each Ala-modified peptide was compared with the natural peptide
sequence.
RESULTS
Optimal amino acid length for microbicidal activity. In or-
der to find an optimal peptide length within the peptide se-
quence 14–31 with regard to antimicrobial activity, constituting
the complete first ␣-helix in the LF molecule, amino acid
residues were removed one by one, starting from the N-termi-
nal end. The reason for keeping the C terminus intact was that
the latter third of the peptide sequence comprised the main
body of positively charged amino acid residues with one Arg at
the C-terminal end (position 31). The antimicrobial activity of
the series of peptides were screened against Enterococcus fae-
calis, Staphylococcus epidermidis, S. aureus, Klebsiella pneu-
moniae, Pseudomonas aeruginosa, E. coli O14, E. coli O6K5,
and C. albicans (Table 1).
The most active peptides with regard to the number of
microbial species or strains that were killed to 99% were the
ones corresponding to residues 17–31, 18–31, 19–31, and 20–31
(Table 1). The trends of increasing antimicrobial activity from
sequence 14–31 to 17–31 and decreasing from sequence 20–31
to 22–31 were statistically significant (chi-square test for
trends) (Table 1).
As shown in the Ala scan (Table 3), the replacement of Asn
by Ala at position 26 in the peptide sequence 20–31 was shown
to increase the antimicrobial activity. Thus, whether any hy-
drophobic or charged amino acid would increase the killing
activity in general when replacing Asn26 or another neutral
hydrophilic amino acid residue in the peptide, such as Gln24,
was investigated. Asn or Gln was replaced by the nonnatural
hydrophobic Nle (norleucine) or positively charged Lys or Orn
(ornithine, a natural amino acid not present in proteins/pep-
tides) (Table 4).
Substitution by Orn or K increased the antimicrobial activity
against E. coli and S. aureus by a four- to eightfold decrease in
the MMC99 value. The Nle substitutions enhanced the killing
activity significantly against S. aureus only (Table 4).
Next, three of the four optimal peptide lengths were ana-
lyzed by twofold serial dilutions in the more concentrated
medium (BP) (Table 2). The most active peptide in BP was
sequence 19–31 (HL9), since this peptide, in contrast to se-
quence 18–31, differed significantly from sequence 20–31. The
killing activity of the peptides increased with time for C. albi-
cans (Table 2).
It should be noted that, in general, replacing BHIdil with
BP reduced the antimicrobial activity of the peptides to
various degrees against E. coli, S. aureus, and C. albicans
(Tables 1 and 2).
(iii) Substitutions by two or more amino acids (Lys, Ala, or
Leu). Increasing the number of positively charged amino acids
by substitution at position 24 (Gln) increased the microbicidal
activity. Combining this replacement with the substitution of
Asn at position 26 by Ala (Table 3) gave rise to a peptide
(CFQWKRAMRKVR, HLopt2) with significantly increased
microbicidal activities for all three microorganisms (Table 4).
Thus, the changes in the peptide increased the activity in BP at
least fourfold against all microbial species. Furthermore, no
significant difference in activity was recorded when comparing
HLopt2 in BP with that in BHIdil (data not shown). Moving the

VOL. 54, 2010
MICROBICIDAL ACTIVITY OF LACTOFERRIN-DERIVED PEPTIDES
421
TABLE 4. Microbicidal activity of modified amino acid sequence
20-31 (CFQWQRNMRKVR)a
TABLE 5. Microbicidal activity of modified HLBD1 peptides
MMC99 ␮g/ml
Amino acid residue
MMC99 ␮g/ml
substitution
Codeb
E. coli
S. aureus
C. albicans
E. coli
S. aureus
C. albicans
Modified HLBD1
Q223A
Positively charged amino acids
Orn or K substitution
Ͼ100
100
12
100
50
25
Ͼ200
37
N263A
CFQWOrnRNMRKVR
CFQWKRNMRKVR
CFQWQROrnMRKVR
25
25
25
50
25
50
25
25
25
Q243K
50
HLBD1
50
50
200
a Peptides were incubated with E. coli O6K5, S. aureus 1800, or C. albicans in
BP. The peptides were diluted in twofold steps, starting at 100 or 200 ␮g/ml (C.
albicans). Each sample was run in duplicate, and the experiment was repeated
once.
Hydrophobic amino acids
Nle substitution
CFQWNleRNMRKVR
CFQWQRNleMRKVR
50
50
50
50
25
50
Two or more substitutions
CFQWKRAMRKVR (HLopt2)
CFAWKRNMRKVR
expressed in the double-sized molecule compared to the native
sequence (Table 5).
12
50
25
25
12
25
50
50
12
25
25
25
CFAWQRAMRKVR
The change of N26 to Ala improved the activity only against
C. albicans, wherein a significant increase of fungicidal activity
was obtained. The increased charge of HLBD1, by changing
Q24 to Lys, improved the microbicidal activity significantly
against both E. coli and C. albicans compared to that of the
natural sequence.
Kinetics of the microbicidal activity. HLBD1 and the short
peptide CFAWKRNMRKVR (Table 4) with corresponding
MMC99 values regarding E. coli and S. aureus were investi-
gated with respect to killing kinetics.
CFALKKAMKKVR
Native sequence
20CFQWQRNMRKVR31
100
Ͼ100
50
a The peptides were incubated with E. coli O6K5, S. aureus 1800 and C.
albicans in BP. The peptides were diluted in 2-fold steps, starting at 100 ␮g/ml.
All samples were run in duplicate. Orn, ornithine, an amino acid not coded for
by DNA and a metabolic intermediate; Nle, norleucine, a nonnatural synthetic
amino acid.
b Substitutions are shown in bold type.
The amount of time needed for killing 90% of E. coli bac-
teria was 1 h, irrespective of the peptide used, when analyzed
at similar concentrations (63 to 65 ␮M) (Fig. 2). While 80% of
S. aureus bacteria were killed within 30 min by HLBD1 (Fig.
2b), the smaller peptide killed 90% within 10 min (Fig. 2a).
More than 90% of C. albicans bacteria were killed within 5 min
by HLBD1 and 99% by the smaller peptide within the same
time span.
Effects of pH and metallic salts on microbicidal activity. (i)
pH. The effects of pH on the antimicrobial activities of HL9,
HLopt2, and HLBD1 were analyzed within the pH range of 4.5
to 7.5 using E. coli and C. albicans (Fig. 3).
HLopt2 was significantly more effective at pHs of 5.5 to 6.5
than at pH 7.0 against both test organisms (fourfold difference
in MMC99) (Fig. 3a and b). For HL9, the microbicidal activity
appeared to increase at pHs of 6.5 to 7.0, regarding E. coli,
while an opposite effect was observed for C. albicans at a pH of
Ն5.5. HLBD1 was significantly less active at pHs lower than
6.5. The MMC99 value increased eightfold at pH 5.5 compared
to that at pH 6.5.
Ala substitution from N26 to Q22 affected the antimicrobial
activity negatively compared with that of the former peptide
sequence (Table 4).
The two separate Ala substitutions that resulted in signifi-
cantly enhanced microbicidal activity (Table 3) were intro-
duced into the same peptide. However, the activity was not
different from that observed by the Ala substitution at N26
(Tables 3 and 4).
In addition to the two Ala substitutions, introducing Lys at
Gln24 and changing the Arg residues at positions 25 and 28 to
Lys also gave rise to a peptide that was significantly better than
the natural one against E. coli and S. aureus.
The only significant enhancing effect that any substitution
(one or several) had on the antimicrobial activity compared
with the natural sequence (20–31), as analyzed in BHIdil, was
obtained with Orn in position 24 against C. albicans (not
shown).
Amino acid substitution in HLBD1. Based on the Ala sub-
stitutions in the peptide sequence 20–31 at Q22 or N26, or to
Lys at Q24 (Tables 3 and 4), the same changes were introduced
in the larger lactoferricin-like HLBD1 (Fig. 1) in order to find
out whether enhanced antimicrobial activities also would be
(ii) NaCl and other metallic salts. At 100 ␮g per ml, both
HL9 and HLopt2 expressed 99% killing activity against E. coli
and C. albicans, up to 50 mM NaCl (Table 6). Doubling the salt
FIG. 1. Amino acid sequence of HLBD1. A synthesized lactoferricin-like cyclic peptide, HLBD1, was based on the first ␣/␤ region of the LF
molecule and comprised amino acid residues 16–40. The full ␣/␤ region of the LF molecule comprises the ␣-helix with amino acid residues 14–31
and a following ␤-sheet with amino acid residues 35–40. The bracket denotes the cyclization by a disulfide bond.

422
HÅVERSEN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Kinetics of the killing activity of HLBD1 and peptide CF
AWKRMRKVR. The peptides were incubated with E. coli strain
O6K5, S. aureus strain 1800, and C. albicans in BP for 20 h using 200
␮g/ml (65.4 ␮M) of HLBD1 and 100 ␮g/ml (62.7 ␮M) of peptide
CFAWKRMRKVR. Samples for viable count were collected directly
after mixing and at 5, 10, 30, 60, and 120 min. All samples were run in
duplicate.
FIG. 3. Effects of various pH levels on the microbicidal activities of
HLopt2, HL9, and HLBD1. All three peptides were analyzed for 99%
killing (MMC99) of E. coli (a), and HLopt2 and HL9 were analyzed for
99% killing of C. albicans (b). The microbicidal assay was run at
various pH levels by adjusting the pH of BP. Twofold serial dilutions
were made of each peptide starting at 100 ␮g/ml of HLopt2 and HL9
and at 200 ␮g/ml for HLBD1. All samples were run in triplicate.
concentration resulted in reduced killing activity. In the pres-
ence of other salts (Kϩ, Mg2ϩ, Ca2ϩ), HLopt2 was consistently
more active at higher concentrations of the metal ions than
HL9, except against C. albicans in the presence of Kϩ, where
both peptides were equally active. Overall, CaCl2 affected the
activity of the peptides the most. HLBD1, which was analyzed
against E. coli at the same concentration as the other two
peptides, was reduced in its activity by all salts to below that of
99% killing (not shown). No salt concentration changed the
viability of the microbial inocula at 2 h of incubation in the
absence of peptides.
comparison of the neutralizing activity of sequence 18–31 or
19–31 with that of HLBD1 showed that the smaller peptides
had significantly stronger inhibitory activities.
The most active peptide, HL9, was further compared with
the known endotoxin-neutralizing agents polymyxin B and BPI
peptide using LPS (Fig. 4b). The two LPSs were blocked in
their Limulus activity to 71% (O111 LPS) and 50% (O55 LPS)
by HL9, a neutralizing effect comparable with those of poly-
myxin B (76 and 51%) and BPI (58 and 61%).
Neutralization of the Limulus activity of LPS and lipid A by
peptide sequences 17–31, 18–31, 19–31 (HL9), and 20–31.
Since one mechanism for the killing of E. coli has been sug-
gested to be mediated via the binding of Lfcin to LPS in the
outer membrane, this binding was studied by a neutralization
test using the Limulus assay. The small peptides, spanning
from 20–31 to 17–31, all neutralized the Limulus activity of
lipid A by 60 to 90% at a concentration of 25 ng/ml (Fig. 4a).
Using the longer peptide, HLBD1, no significant neutralizing
effect was obtained for the diphosphoryl lipid A, while approx-
imately 50% of the monophosphoryl lipid A was neutralized. A
DISCUSSION
The complete antimicrobial mechanism of action of LF and
peptides derived from it is unknown, although considerable

VOL. 54, 2010
MICROBICIDAL ACTIVITY OF LACTOFERRIN-DERIVED PEPTIDES
423
TABLE 6. Effects of metallic salts on the antimicrobial activities of
ular to the core (20) when interacting with LPS. The additional
Cys-Lys at the N-terminal end in HL9 may facilitate or stabi-
lize a conformation of the amphipathic peptide, which relies on
three clusters of charged amino acids. Another possible con-
formation of HL9 includes a central ␤-strand with charged
amino acids at the ends, as was proposed for a corresponding
sequence of the 11-mer peptide (residues 20-30) of bovine LF
(12). Irrespective of conformational shape, the chain size of
HL9 appears to be optimal for conformation plasticity while
still retaining the ability to form a defined tertiary structure
in the presence of bacterial membranes. It should be noted
that random conformations of the peptides are present in
solution. Additionally, the presence of Lys in HL9 fits the
hypothesis that positively charged amino acids add to anti-
microbial activity.
The alanine scan of the shortest peptide sequence (20-31)
with retained killing activity (Table 1) showed that single-
amino-acid substitutions did not result in total abolishment of
microbicidal activity. However, considerable reduction in an-
tifungal activity was observed when Ala was substituted for
Cys, Trp, or the charged amino acid clustered at the C-terminal
end of the peptide (positions 28, 29, and 31) with regard to C.
albicans, a pattern that fits the T-shaped conformational
model, since Trp was found to be the central residue of the
hydrophobic core and the additional Cys20 could possibly en-
HL9 and HLopt2 against E. coli O6K5 and C. albicans 1800a
99% killing activityb
E. coli C. albicans
HLopt2
Salinity or
metallic salt
mM
HL9
HL9
HLopt2
Salinity
NaCl
25
50
100
ϩ
ϩ
Ϫ
ϩ
ϩ
Ϫ
ϩ
ϩ
Ϫ
ϩ
ϩ
Ϫ
NaCl
NaCl
Metallic salts
KCl
25
50
100
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
ϩ
ϩ
Ϫ
KCl
KCl
MgCl2
MgCl2
MgCl2
1
ϩ
Ϫ
Ϫ
ϩ
ϩ
Ϫ
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
2.5
5.0
CaCl2
CaCl2
CaCl2
1
2.5
5.0
Ϫ
Ϫ
Ϫ
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
Ϫ
Ϫ
a Both peptides were analyzed at a concentration of 100 ␮g/ml. The salts were
added to BP. All samples were run in triplicate.
b ϩ, Ն99% killing of the inoculum; Ϫ, Ͻ99% killing.
efforts toward understanding the relationship between struc-
ture and antimicrobial activity from studies of LF and LF
peptide fragments have shed light on parts of their functions.
We have previously shown that, in addition to LF, HLBD1 and
a similar peptide (amino acid residues 18–40), based on the
first ␣-helix–␤-sheet region of the LF molecule, are efficient in
reducing infection and inflammation in vivo in experimental E.
coli-induced urinary tract infections and colitis (16, 17).
Peptides other than pepsin-derived lactoferricin may occur
locally at mucosal surfaces or in tissues where various pro-
teases may digest LF differently to as-yet-unknown peptide
fragments. The antimicrobially active region in lactoferricin
also included the first cationic domain of human LF compris-
ing amino acid residues 1–11. This short 11-amino-acid-long
peptide and the recently reported peptide comprising residues
153–183 of the LF molecule also have been shown to express
antimicrobial activities (11, 26, 30, 32).
It is not clear whether the intact LF protein or peptides
derived from it by proteolytic cleavage have the most active
LF-induced antimicrobial activity in vivo.
In the present study, the peptide fragment 14-31, corre-
sponding to the first ␣-helix of the LF molecule, was downsized
by removing one amino acid at a time from the N-terminal end.
A 13-amino-acid-long peptide (19-31, HL9) was found to be
the most active with respect to the overall antimicrobial effects.
Downsizing beyond residues 20-31 resulted in a considerable
loss of activity. These results suggested that Lys19 and Cys20 at
the N terminus were important for optimal killing activity of
the natural sequence.
It is interesting to note that an amino acid sequence (Met at
position 27 changed to Ile) almost identical to our antimicro-
bial peptide sequence 21-31, which was significantly weaker
than HL9 or peptide sequence 20-31, was reported to adopt a
T-shaped arrangement of a hydrophobic core composed of the
N-terminal end and two clusters of basic residues perpendic-
FIG. 4. Neutralization of the Limulus activities of lipid A (a) and
LPS (b) by the peptide sequences containing amino acid residues
20–31 to 17–31. (a) The peptides (25 ng/ml) of amino acid residues
20–31 to 17–31 were incubated with diphosphoryl and monophospho-
ryl lipid A, and the remaining Limulus activity was analyzed (expressed
as percentage of maximal LAL activity). The experiment was repeated
three times, except for peptide sequences 17–31 and 20–31 (single run
for diphosphoryl lipid A; repeated once for monophosphoryl lipid A).
, P Ͼ 0.05; , P Ͼ 0.01 (Student’s t test). In panel b, the remaining
*
**
activity of LPS O111 and O55 after incubation with 5 ␮g/ml of peptide
sequence 19–31 (HL9), polymyxin B, and BPI peptide is shown. The
experiment was repeated once except for O111 LPS (single run).

424
HÅVERSEN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
hance the hydrophobic core (20). In general, Trp is important
for its ability to cluster hydrophobic contacts around its side
chain (8, 21). The importance of Trp and Arg residues for
antimicrobial activity has also been shown for bovine Lfcin,
residues 20-29 of bovine LF, as well as other synthetic antimi-
crobial peptides (7, 13, 29, 33, 36). The replacement of any of
the charged amino acids also corresponded with weakened
charged clusters of the upper part of the T shape. Unlike in
bacteria, the cytoplasmic membranes of yeast cells contain the
unique lipid ergosterol, and the cell wall is composed of chitin
and ␤-glucan with an outer layer of mainly phosphate-contain-
ing mannoproteins. Thus, it may not be unexpected to find
various patterns of antimicrobial activity.
six residues at the N terminus of the 33-mer peptide appeared
critical for the antiendotoxin activity. Our results, however,
indicated that the shorter peptides, comprising 12 to 15 amino
acids and lacking the six N-terminal residues, retained endo-
toxin-neutralizing activity and were comparable with the well-
known antiendotoxic agents polymyxin B and BPI peptide (24).
Our results are in agreement with those of others, wherein the
peptides containing residues 21-31 and another peptide very
similar to residues 21-31 were found to bind to LPS (5, 12, 13,
20). The strongest interaction was obtained with HL9.
The differences between E. coli, S. aureus, and C. albicans,
with respect to the kinetics of killing by short peptide, most
probably reflect the difference in the construction of their cell
envelopes. In two reports using synthetic peptides based on the
residues 21-31 or 16-40, it was suggested that binding to LPS
may be an initial event leading to penetration of the outer
membranes, followed by interaction with the cytoplasmic
membranes, of E. coli (1, 6). The peptide corresponding to
residues 18-40 in LF was shown to interact with the cytoplas-
mic membranes of E. coli and cause losses of the bacterial
transmembrane electrical potential and the pH gradient (1,
35). Using E. coli O111, Chapple et al. observed a time-depen-
dent collapse of the membrane potential and membrane integ-
rity upon exposure to peptide sequence 21-31. In agreement
with our study, a lag phase of 1 to 2 h, depending on the
peptide concentration, was needed for the killing of E. coli (5,
6). Our observation of binding of the peptides to LPS and lipid
A supports a model in which the first step includes the inter-
action with LPS. This, together with the suggestion that the
peptide folds and self-assembles at the outer membrane sur-
face, indicates a two-stage process of the antibacterial activity
against E. coli and could explain the longer time needed for the
peptide to exert its activity against this bacterium compared to
that against S. aureus and C. albicans (5).
For E. coli killing activity, the most important residue was
Cys, since replacement by Ala reduced the antibacterial activ-
ity significantly.
Modifications of peptide sequence 20-31 by increasing the
number of positively charged or hydrophobic amino acids en-
hanced the microbicidal activity in general. Even nonnatural
building blocks of peptides, such as the hydrophobic Nle or
basic Orn, enhanced the antimicrobial activity. Possibly, such
amino acids could increase the resistance to degradation of the
peptide. Thus, substituting neutral hydrophilic amino acids,
such as Asp and Gln, by either single or double substitution
(HLopt2) improved the microbicidal activity four- to eightfold
in BP, resulting in activities close to those obtained in diluted
BHI. Furthermore, comparing the modified HLopt2 with its
corresponding unmodified form or the longer HL9 showed
that the fungicidal activity was enhanced more than four- and
10-fold, respectively. Additionally, single-amino-acid substitu-
tions in the double-sized HLBD1 revealed that exchange of
Gln24 to Lys enhanced the killing activity significantly against
E. coli and C. albicans compared to those of the unmodified
peptide (Table 5) and similar levels of HLopt2 for E. coli and
S. aureus (Table 4).
With regard to the kinetics of the microbicidal activity to-
wards S. aureus, the smaller size of the peptide appeared to
increase the rate of killing.
Regarding the effects of salts, it is known that LF peptides,
in accordance with ␣ and ␤ defensins, are sensitive to high-salt
environments (14, 28). Thus, an ionic strength of 100 mM NaCl
weakened the lethal activity of our peptides. The strongest
The rapid killing of C. albicans by both peptides may indi-
cate that there is a somewhat different mode of action and that
the cell wall is easier to pass through. A synthetic fragment of
the first cationic domain in the LF molecule from the N-
terminal end comprising amino acid residues 1-11 was sug-
gested to interact with structural elements of the plasma mem-
brane of C. albicans and be taken up in an energy-dependent
way. The following induced release of ATP, combined with the
activity of the peptide, was essential for the candidacidal ac-
tivity (22). A peptide corresponding to amino acid residues
17-26 from bovine LF has also been shown to enhance the
candidacidal activity of antifungal drugs by inducing the ATP
efflux from C. albicans (31). In a following paper, it will be
shown that mitochondrial membranes are affected in the kill-
ing process.
In conclusion, the residue fragment 19-31 comprising more
than half of the helix in the LF molecule was the most active
microbicidal fragment against E. coli, S. aureus, and C. albi-
cans. The Cys at the N-terminal end (position 20), a hydro-
phobic residue at position 23, and the charged amino acids at
positions 28 and 31 were important for antimicrobial activity.
However, replacement of the polar amino acid in the shorter
peptide sequence 20-31 at positions Q24 by Lys and N26 by
influence on the microbicidal activity was observed with Ca2ϩ
.
Clearly, the ionic environment affects the microbicidal activity
in a complex way, also including effects on the target microor-
ganism. Regarding the pH dependence, the lethal activity of
HLopt2 was unaffected at pHs of 4.5 to 6.5, while HL9 showed
no significant changes within the tested pH range (pHs of 4.5
to 7.5). In contrast, the lethal effect of the longer cyclic peptide,
HLBD1, was impaired at pHs below 6.5 (analyzed only for E.
coli). Thus, the pattern of pH dependence appeared to be
specific for each peptide.
By neutralization of the endotoxic activity measured by the
Limulus assay, we showed that the short peptides (17-20-31)
bound to both lipid A and LPS. The effect may be exerted by
binding partly to different binding sites on lipid A and LPS,
respectively. Both phosphate and carboxyl groups are available
on the LPS molecule, but only phosphates on the lipid A
molecule. The neutralization of endotoxin activity in vitro by
the Limulus assay and by suppression of endotoxin-induced
tumor necrosis factor alpha secretion in macrophages and in
vivo by lethal endotoxin challenge in mice has been shown with
a peptide corresponding to residues 1-33 of LF (38). The first

VOL. 54, 2010
MICROBICIDAL ACTIVITY OF LACTOFERRIN-DERIVED PEPTIDES
425
18. Håversen, L. A., I. Engberg, L. Baltzer, G. Dolphin, L. A. Hanson, and I.
Mattsby-Baltzer. 2000. Human lactoferrin and peptides derived from a sur-
face-exposed helical region reduce experimental Escherichia coli urinary
tract infection in mice. Infect. Immun. 68:5816–5823.
19. Hunter, H. N., A. R. Demcoe, H. Jenssen, T. J. Gutteberg, and H. J. Vogel.
2005. Human lactoferricin is partially folded in aqueous solution and is
better stabilized in a membrane mimetic solvent. Antimicrob. Agents Che-
mother. 49:3387–3395.
20. Japelj, B., P. Pristovsek, A. Majerle, and R. Jerala. 2005. Structural origin of
endotoxin neutralization and antimicrobial activity of a lactoferrin-based
peptide. J. Biol. Chem. 280:16955–16961.
21. Klein-Seetharaman, J., M. Oikawa, S. B. Grimshaw, J. Wirmer, E. Duch-
ardt, T. Ueda, T. Imoto, L. J. Smith, C. M. Dobson, and H. Schwalbe. 2002.
Long-range interactions within a nonnative protein. Science 295:1719–1722.
22. Lupetti, A., A. Paulusma-Annema, M. M. Welling, S. Senesi, J. T. van Dissel,
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44:3257–3263.
Ala not only significantly reduced the MMC99 values in com-
parison to the natural sequence but led to the most active
peptide of all the peptides analyzed.
ACKNOWLEDGMENTS
The excellent technical assistance of Lotta Arna¨s is gratefully
acknowledged.
This study was financially supported by Aϩ Science Invest AB,
Gothenburg, Sweden.
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