In various vertebrates, neutralization of the endotoxin, LPS complex, of the outer membranes of gram-negative bacterial pathogens is important for protection against excessive inflammation during bacterial infections of a variety of tissues [1, 2]. The most significant LPS neutralization mechanism in human blood is the acute phase protein named LBP , whereas related antimicrobial peptides operate in other tissues . Both LBP and antimicrobial peptides interfere with the proinflammatory 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 proinflammatory and chemotactic cytokines interleukin-1, interleukin-6 and tumor necrosis 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 insufficient amounts of these factors is associated with dysregulation and overrated responses leading to intensive tissue inflammation and damage and endotoxic shock .
Although LPS induces inflammatory responses in ectothermic 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 DNA in two frog species, Xenopus laevis and Silurana tropicalis , its protein product has not yet been identified 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 [8-10]. These peptides have positively charged amino acid sequences that are required for interaction with negatively charged components of microbial membranes and lysis of microbial cells . 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 . Moreover, researchers have also demonstrated the ability of skin antimicrobial peptides to bind to LPS from 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 purified it by using C18-Sep-Pak cartridges (Waters Corporation, Milford, MA, USA) as described by Schadich . We confirmed its content of species-specific aurein peptides by liquid chromatography mass spectrometry analyses . Polymixin B sulfate, a reference control peptide with known ability to neutralize endotoxins from gram-negative bacteria and endotoxin-free 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 37°C for 20 hrs. After digestion, we inactivated the protease by heating it at 90°C for 10 min.
Using a modified phenol–water technique as previously described , 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 blood-agar 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 (QCL-1000 kit, Cambrex Bio Science). We sterilized all pyrogenic-free consumables by heating them for 3 hrs at 180°C.
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. . We incubated the peptides dissolved in endotoxin-free water at different concentrations of peptide (0–300 μg/mL) with 150 pg/mL of bacterial LPS in 50 μL reactions at 37°C for 30 min. The blank controls included the same concentration without the LPS. Subsequently, we incubated 50 μL of the LAL reagent containing pro-enzyme at 37°C for 10 min in 96-well microtiter plates. Next, we added 100 μL of the LAL substrate to each sample and incubated them at 37°C for an additional 6 min. We stopped the reactions using 100 μL 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 IC50 values of peptides (peptide concentration at which 50% of LPS is neutralized) 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 IC50 values for neutralization of LPS of E. coli (0111:B4) and K. pneumoniae were below 100 µg/mL (Table 1). The peptide digests did not neutralize any of two bacterial LPSs.
Figure 1. Neutralization of (a) Escherichia coli (0111:B4) and (b) Klebsiella pneumoniae lipopolysaccharide (LPS) by skin antimicrobial peptides of Litoria raniformis frogs and polymyxin B.
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Table 1. Lipopolysaccharide neutralization activity of skin antimicrobial peptides of Litoria raniformis and polymixin B
|LPS||IC50 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|
Skin antimicrobial peptides of L. raniformis neutralized the LPS from bacterial pathogen K. pneumoniae in vitro at physiologically relevant concentrations (IC50 < 100 μg/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 non-specific 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.