NOTE

Neutralization of bacterial endotoxins by frog

antimicrobial peptides
Ermin Schadich, Drusilla Mason and Anthony L. Cole
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
ABSTRACT
The ability of skin antimicrobial peptides of the southern bell frog, Litoria raniformis, to neutralize in
vitro the endotoxin, proinammatory lipopolysaccharide (LPS) complex, from two different gram
negative bacterial pathogens, human pathogen Escherichia coli (0111:B4) and frog pathogen Klebsiella
pneumoniae, was investigated. The LPS neutralization activity of the natural mixture of skin anti-
microbial peptides was measured using chromogenic Limulus amebocyte lysate assays. These skin
antimicrobial peptides neutralized the LPSs from both pathogens at physiologically relevant
concentrations (IC
50
< 100 g/mL) showing their potential for nonspecic LPS neutralization
in vivo in the skin of infected frogs and for development of antiendotoxin agents.
Key words antimicrobial peptides, bacterial lipopolysaccharide, inflammation.
In various vertebrates, neutralization of the endotoxin,
LPS complex, of the outer membranes of gramnegative
bacterial pathogens is important for protection against
excessive inammation during bacterial infections of a
variety of tissues (1, 2). The most signicant LPS
neutralization mechanism in human blood is the acute
phase protein named LBP (3), whereas related antimi-
crobial peptides operate in other tissues (4). Both LBP
and antimicrobial peptides interfere with the proin-
ammatory effects of LPSs on macrophages by binding
to its lipid component and thereby precluding its
interaction with TLR4 and associated pathways of
production of the proinammatory and chemotactic
cytokines interleukin1, interleukin6 and tumor necro-
sis factor (3, 4). They are essential for regulation of
subsequent neutrophil responses and activation of the
complement lytic system. Any pathogenic process that
results in insufcient amounts of these factors is
associated with dysregulation and overrated responses
leading to intensive tissue inammation and damage and
endotoxic shock (2).
Although LPS induces inammatory responses in ecto-
thermic vertebrates like frogs, it has little toxicity in these
animals (5, 6). Surprisingly, although the sequences of
genes orthologous to the human LPB have been found in
genomic DNAintwo frog species, Xenopus laevis and Silurana
tropicalis (7), its protein product has not yet been identied
either in blood or liver. This is intriguing as it raises the
question of how frogs protect themselves from LPS.
One possible LPS neutralization mechanism in frogs
is antimicrobial peptides from skin granular glands,
stomach and intestinal tissues (810). These peptides
have positively charged amino acid sequences that are
required for interaction with negatively charged compo-
nents of microbial membranes and lysis of microbial
cells (11). A previous study by Schadich showed that
antimicrobial peptides from skin granular glands of
different frog species have activity against different
human and frog bacterial pathogens, an activity which
strongly correlates with resistance to bacterial disease
(12). Moreover, researchers have also demonstrated the
ability of skin antimicrobial peptides to bind to LPS from
Correspondence
Ermin Schadich, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, NewZealand. Tel: 64 3 364 2500; fax: 64
3 364 2590; email: ermins@gmail.com
Received 28 September 2012; revised 11 November 2012; accepted 25 November 2012.
List of Abbreviations: E. coli, Escherichia coli; K. pneumoniae, Klebsiella pneumoniae; IC
50
, values of peptides peptide concentration at which 50%of
LPS is neutralized; LAL, Limulus amebocyte lysate; LBP, lipopolysaccharidebinding protein; LPS, lipopolysaccharide; L. raniformis, Litoria raniformis ;
TLR4, Tolllike receptor 4.
Microbiol Immunol 2013; 57: 159161
doi: 10.1111/1348-0421.12012
2012 The Societies and Wiley Publishing Asia Pty Ltd 159
different human bacterial pathogens (13, 14), and this
suggests their possible role in the LPS neutralization
mechanism.
Skin antimicrobial peptides could therefore provide
protection from the toxic effects of endotoxins in frog
skin. This study aimed to determine whether skin
antimicrobial peptides of the New Zealand introduced
species, the southern bell frog (L. raniformis) can neutralize
LPS from different bacterial pathogens in vitro. We tested
skin antimicrobial peptides from L. raniformis for their
ability to neutralize LPS from the human pathogen E. coli
(0111:B4) and the frog pathogen K. pneumoniae.
We collected a natural mixture of skin antimicrobial
peptides from adult L. raniformis by using norepinephrine
injections and partially puried it by using C18SepPak
cartridges (Waters Corporation, Milford, MA, USA) as
described by Schadich (12). We conrmed its content of
speciesspecic aurein peptides by liquid chromatogra-
phy mass spectrometry analyses (12). Polymixin Bsulfate,
a reference control peptide with known ability to
neutralize endotoxins from gramnegative bacteria and
endotoxinfree water were purchased from Sigma
Chemical (St. Louis, MO, USA). We generated peptide
digests to provide a negative control for studies of the
activity of the skin peptides. We incubated the peptide
mixtures (1 mg/mL) with pronase E, a protease mixture
that degrades peptides completely (Sigma Chemical), at a
concentration of 0.5 mg/mL in ammonium phosphate
buffer (pH 7.0) at 37C for 20 hrs. After digestion, we
inactivated the protease by heating it at 90C for 10 min.
Using a modied phenolwater technique as previ-
ously described (15), we isolated LPS from overnight
colonies of K. pneumoniae collected from the wild brown
tree frog, Litoria ewingii in Oxford forest, Zealand, and
grown on bloodagar plates. We purchased the LPS of E.
coli (0111:B4) from Cambrex Bio Science (Walkersville,
MD, USA). We tested all solutions to ensure they were
endotoxin free by measuring the concentration of LPS
using chromogenic LAL assays (QCL1000 kit, Cambrex
Bio Science). We sterilized all pyrogenicfree consum-
ables by heating them for 3 hrs at 180C.
We assessed neutralization of LPS by skin antimicrobial
peptides by measuring their free concentrations using
LAL assays after incubating them with skin peptides as
described by Ried et al. (16). We incubated the peptides
dissolved in endotoxinfree water at different concen-
trations of peptide (0300 mg/mL) with 150 pg/mL of
bacterial LPS in 50 mL reactions at 37C for 30 min. The
blank controls included the same concentration without
the LPS. Subsequently, we incubated 50 mL of the LAL
reagent containing proenzyme at 37C for 10 min in 96
well microtiter plates. Next, we added 100 mL of the LAL
substrate to each sample and incubated them at 37C for
an additional 6 min. We stopped the reactions using
100 mL of 25% acetic acid and read the absorbance of
each reaction at 405 nm using a microplate reader. We
used the absorbance values for curve analyses. We
performed three assays for both tested peptides and
controls and tested three replicate reactions for each
peptide concentration. We automatically adjusted all
curves required for estimation of IC
50
values of peptides
(peptide concentration at which 50% of LPS is neutral-
ized) by nonlinear regression using Graph Pad Prism 4.
The peptide mixture from L. raniformis neutralized the
LPS of the standard reference strain E. coli (0111:B4) and
the isolate K. pneumoniae in a concentration dose
dependent manner comparable to the reference control
polymyxin B (Fig. 1). Their IC
50
values for neutralization
of LPS of E. coli (0111:B4) and K. pneumoniae were below
Fig. 1. Neutralization of (a) Escherichia coli (0111:B4) and (b)
Klebsiella pneumoniae lipopolysaccharide (LPS) by skin anti-
microbial peptides of Litoria raniformis frogs and polymyxin B.
E. Schadich et al.
160 2012 The Societies and Wiley Publishing Asia Pty Ltd
100 g/mL (Table 1). The peptide digests did not
neutralize any of two bacterial LPSs.
Skin antimicrobial peptides of L. raniformis neutralized
the LPS from bacterial pathogen K. pneumoniae in vitro at
physiologically relevant concentrations (IC
50
< 100 mg/
mL; Table 1), suggesting that they may be an LPS
neutralization mechanism in infected frogs. This activity
is not restricted to frog pathogens since the LPS from
human pathogen E. coli (0111:B4) was also neutralized
(Fig. 1, Table 1). Such nonspecic in vitro activity could
be an effective, broad and rapid mechanism for
neutralization of LPS from different bacterial pathogens
in vivo in the skin of infected frogs.
The peptide mixture of L. raniformis was not as active as
polymixin B in neutralizing LPS; this may have been due
to dilution of the mixture by inactive peptides. Thus, one
direction of future studies should be also to analyze the
effects of single isolated peptides in order to show their
potential in LPS neutralization.
ACKNOWLEDGMENTS
We thank Andrew Bagshaw, University of Otago for
useful comments on this manuscript. This study was
supported by a Royal Society of New Zealand Marsden
Grant (M1069).
DISCLOSURE
All authors have no conict of interest.
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Table 1. Lipopolysaccharide neutralization activity of skin antimicro-
bial peptides of Litoria raniformis and polymixin B
LPS
IC
50
of peptides
L. raniformis Polymixin B
E. coli (0111:B4) 64.5 1.1 2.0 1.2
K. pneumoniae 55.8 1.2 1.7 1.0
2012 The Societies and Wiley Publishing Asia Pty Ltd 161
LPS neutralization by frog peptides