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Antimicrobial Agents and Chemotherapy, May 1999, p. 1267-1269, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
N-Acylated and D Enantiomer Derivatives
of a Nonamer Core Peptide of Lactoferricin B Showing Improved
Antimicrobial Activity
Hiroyuki
Wakabayashi,1,*
Hiroshi
Matsumoto,2
Koichi
Hashimoto,2
Susumu
Teraguchi,1
Mitsunori
Takase,1 and
Hirotoshi
Hayasawa1
Nutritional Science
Laboratory1 and Biochemical Research
Laboratory,2 Morinaga Milk Industry Co.,
Ltd., Zama, Kanagawa 228-8583, Japan
Received 2 September 1998/Returned for modification 8 January
1999/Accepted 7 March 1999
 |
ABSTRACT |
N-acylated or D enantiomer peptide derivatives based on
the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and
fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl
group, showed two to eight times lower MIC than lactoferricin B.
| |
TEXT |
Lactoferricin B (LFcin B) is a
25-mer antimicrobial peptide generated by pepsin digestion of bovine
lactoferrin (LF) and is more potent than lactoferricin H (LFcin H), a
47-mer peptide similarly derived from human LF (3). LFcin B
shows activity against a wide range of microorganisms including
bacteria and fungi (2, 15), and its mode of action is
microbicidal via membrane perturbation (4, 15). The peptide
shows synergistic actions with other antimicrobial agents such as azole
antifungal agents (13, 14). Nuclear magnetic resonance
studies have revealed that the three-dimensional structure of LFcin B
is an antiparallel 
-sheet and that hydrophilic and positively
charged residues surround the hydrophobic surface, suggesting
interactions of this peptide with the biological membrane (9). Although several LFcin analogs with amino acid
sequences shorter than LFcin B or LFcin H have been reported, their
antimicrobial potency seems to be similar to or weaker than that of the
original LFcin (5, 7, 10, 11). Therefore, this study was
performed to produce more potent LFcin analogs and demonstrate improved antimicrobial activities displayed by N-acylated or D
enantiomer analogs of nonamer peptides based on a core sequence of
LFcin B.
Preparation of peptides.
The structures of the LFcin-related
peptides and reference antimicrobial peptides used in this study are
shown in Table 1. LFcin B was purified
from a pepsin hydrolysate of bovine LF as described previously
(3). (Ala8,13,18)-magainin II-NH2
(6) was purchased from Sigma Chemical Co. (St. Louis, Mo.).
Other peptides were chemically synthesized on a
4-(2',4'-dimethoxyphenyl-9-fluorenylmethoxycarbonyl-aminomethyl) phenoxyacetamido-ethyl resin or preloaded
4-(hydroxymethyl)phenoxymethyl-copoly(styrene-1% divinylbenzene)
(HMP) resin (Perkin-Elmer Japan Co. Applied Biosystems Division, Chiba,
Japan) by a 9-fluorenylmethoxycarbonyl strategy and solid-phase
methodology by means of an automated synthesizer (model 433A; Perkin
Elmer). For N-acylation of the peptides, the resin was treated with the
desired fatty acid activated in situ as an HOBt/HBTU ester. After
removal of the resin and the side-chain-protecting groups, the crude
peptides were purified by reverse-phase high-performance liquid
chromatography. The structures of the final compounds were confirmed by mass-spectral analysis (LCQ, Finnigan Mat). All peptides including N-acylated ones were soluble in water.
Microorganisms tested.
Escherichia coli IID861,
Pseudomonas aeruginosa IFO3445, and Staphylococcus
aureus JCM2151 (ATCC 6538P) were maintained on plate count agar
(Eiken Chemicals Co., Tokyo, Japan) slants. The stock cultures were
harvested in 1% Bacto Peptone (Difco Laboratories Co., Detroit, Mich.)
and incubated for 7 h at 37°C to obtain inocula. Candida
albicans TIMM0144 and Trichophyton mentagrophytes
TIMM1189 were grown on Sabouraud glucose agar (1% Bacto Peptone, 2%
glucose, 1.5% agar) slants at 27°C before use.
Antimicrobial susceptibility testing.
MICs were determined by
the microdilution method using flat-bottom 96-well microplates. As
culture media, 1% Bacto Peptone or Mueller-Hinton broth (Difco) was
used for bacteria and Sabouraud glucose broth (1% Bacto Peptone, 2%
glucose) was used for fungi. Bacterial cells were inoculated into
microplates at a final cell density of 106 cells/ml and
incubated for 17 h at 37°C. C. albicans cells were harvested from a slant, suspended in the culture medium, inoculated into microplates at 105 cells/ml, and incubated for 17 h at 37°C. Microconidia of T. mentagrophytes were
harvested from a slant in saline solution containing 0.85% NaCl and
0.05% Tween 80, suspended in the culture medium, inoculated into
microplates at 104 cells/ml, and incubated for 4 days at
27°C. The MIC for triplicate samples was determined.
The MICs of LFcin-related peptides for the five microorganisms in
peptone-based broth are shown in Table
2.
LFcin H(25mer),
which has an amino acid sequence homologous to that of
LFcin B,
was substantially less active than LFcin B against all
microorganisms
tested. We synthesized several peptide derivatives based
on the
cationic-amino-acid-rich region RRWQWRMKK (corresponding to
positions
R4 through K12) of LFcin B and assayed the antimicrobial
activity
of each. N-acyl derivative peptides having a carbon chain
length
of six to eleven carbons showed similar or higher activities
compared
with LFcin B. The most active peptide had an 11-carbon acyl
chain
and exhibited two- to four-times-lower MIC than LFcin B.
D-(4-12)-NH
2,
a
D enantiomer
derivative of (4-12)-NH
2, was also highly active
and showed
MIC similar to that of Acyl-11-(4-12)-NH
2. N-acylation,
however, did not further augment the activity of the
D
enantiomeric
peptide. Ac-RRWWCR-NH
2 was previously
identified as the most potent
hexameric antimicrobial peptide in
synthetic peptide combinatorial
libraries (
8) and showed
sequence similarity to the RRWQWR
region in LFcin B. This peptide
showed slightly lower activity
than LFcin B. (Ala
8,13,18)-magainin II-NH
2 is a peptide
consisting of 23 amino acid residues
reported to display antimicrobial
activity up to two orders of
magnitude stronger than that of the
original magainin II (
6).
The activity of this analog was
similar to that of Acyl-11-(4-12)-NH
2.
In tests using
Mueller-Hinton broth, in which the activity of
LFcin B, especially
against gram-negative bacteria, was decreased,
improvement of
antimicrobial activities and carbon chain length-dependent
activities
for N-acylated analogs were clearly indicated (Table
3). Acyl-10-(4-12)-NH
2 and
Acyl-11-(4-12)-NH
2 showed four- to
eight-times-lower MIC
than LFcin B.
N-acylation resulted in an increase in the interaction of LFcin B
analogs with microbial membranes and may have caused enhanced
antimicrobial activity. Appropriate amino acid substitution is
known to
increase the activity of antimicrobial peptides (
6,
10). The
N-acyl derivatization approach using a relatively short
peptide,
however, can simplify the peptide synthesis process compared
to
synthesizing larger peptides. Although Ca
2+ or
Mg
2+ ions (>2 mM) are known to reduce the activity of
LFcin B (
2),
these ions were not likely involved in the
increased MIC of the
peptide in Mueller-Hinton broth, because the
concentrations of
Ca
2+ and Mg
2+ in
Mueller-Hinton broth were low (0.15 and 0.21 mM, respectively)
and
similar to those in 1% Bacto Peptone. Other constituents may
be
responsible for the diminished activity of LFcin B in Mueller-Hinton
broth, and N-acylated derivatives may overcome such blocking effects
of
medium ingredients.
D enantiomer peptides can survive
digestion
by proteases secreted by microorganisms. This and previous
studies
(
1,
5,
12) indicate advantages of
D
enantiomer derivatives
in antimicrobial
applications.
The hemolysis activity of the 11-mer LFcin B analog is reported to be
very low (
10), and
D-(4-12)-NH
2 did
not exert a toxic
effect against animal cells at concentrations active
for the inhibition
of microorganisms in our preliminary evaluation.
Oral application
is a possible route for the therapeutic uses of the
derived compounds,
because oral administration of the N-acylated and
D enantiomer
derivatives in mice resulted in no lethal
toxicity at doses as
high as 100 mg/kg of body weight (unpublished
data).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Nutritional
Science Laboratory, Morinaga Milk Industry Co., Ltd., 5-1-83 Higashihara, Zama, Kanagawa 228-8583, Japan. Phone: 81-462-52-3045. Fax: 81-462-52-3049. E-mail: Hiroyuki_Wa{at}msn.com.
| |
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Antimicrobial Agents and Chemotherapy, May 1999, p. 1267-1269, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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