Comparative Biochemistry and Physiology, Part B 158 (2011) 149–154

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Comparative Biochemistry and Physiology, Part B

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Isolation and identification of pelteobagrin, a novel antimicrobial peptide from the

skin mucus of yellow catfish (Pelteobagrus fulvidraco)
Yueju Su ⁎
School of Animal Science and Technology, Jinling Institute of Technology, Nanjing 211169, China

a r t i c l e i n f o a b s t r a c t

Article history: Fish skin mucus has recently been recognized to be a potential source of antimicrobial peptides, which
Received 26 July 2010 provides the first line of defense against invading pathogens. This study reports the purification and
Received in revised form 2 November 2010 characterization of a novel linear antimicrobial peptide, pelteobagrin, from the skin mucus of yellow catfish
Accepted 3 November 2010
(Pelteobagrus fulvidraco, Richardson). Pelteobagrin is 20 amino acids in length (GKLNLFLSRLEILKLFVGAL) and
Available online 10 November 2010
shows no clear homology with any known bioactive peptides. MALDI-TOF MS indicated the molecular mass of
the purified peptide was 2244.4 Da, which is in good agreement with pelteobagrin's predicted molecular
Keywords:
Antimicrobial peptide
weight of 2244.8 Da. Pelteobagrin exhibited antibacterial activity against Gram-positive and Gram-negative
Hemolytic bacteria as well as fungi, and the activity was relatively salt-insensitive as it was not affected by NaCl
Mesosome concentrations of up to 137 mM. Moreover, pelteobagrin displayed no hemolytic activity to rabbit red blood
Transmission electron microscopy cells. Transmission electron microscopy suggested that pelteobagrin might kill bacteria via acting on both the
cell wall and the cytoplasmic membrane of bacteria. These results suggest that pelteobagrin might be involved
in the innate defense system in yellow catfish.
© 2010 Elsevier Inc. All rights reserved.

1. Introduction first line of protection against water-borne microbes. Besides function-

ing as a physical barrier between fish and its aquatic environment, the
Antimicrobial peptides (AMPs), also called host defense peptides, skin mucus also has been demonstrated to have antimicrobial activity
are an evolutionarily conserved component of the innate immune mediated via an array of innate immune factors such as lysozyme,
system and are found among all classes of life. AMPs are generally lectins, proteolytic enzymes, flavoenzymes, immunoglobulins, and C-
between 10 and 50 amino acids with a net positive charge of 2 to 8. reactive protein (Ellis, 2001; Whyte, 2007; Subramanian et al., 2008).
These peptides exhibit a wide range of activity against numerous More recent investigations have revealed that the surface mucosa of
microbes including Gram-positive and Gram-negative bacteria, fungi, some fish also contains a range of AMPs (Oren and Shai, 1996; Cole et al.,
viruses, and parasites while with little or no toxicity to host cells 1997; Park et al., 1998b; Birkemo et al., 2003; Fernandes et al., 2003;
(Hancock and Scott, 2000). In addition, certain peptides have the Fernandes et al., 2004; Bergsson et al., 2005; Lüders et al., 2005;
ability to enhance immunity by functioning as immunomodulators Subramanian et al., 2009).
(Bowdish et al., 2005). To date, about 1200 AMPs have been In an attempt to identify novel antibacterial peptides that might
characterized, and a large number of them were isolated from mediate innate immunity in fish, the skin mucus from several
mammals, amphibians, insects or other invertebrates. Although fish commercially available fish species was examined for the antimicro-
are by far the most abundant vertebrate in the world, relatively few bial activity, with the skin mucus of yellow catfish (Pelteobagrus
AMPs have been isolated from fish presumably reflecting a simple lack fulvidraco, Richardson) showing the strongest antimicrobial activity.
of attention to this potentially rich source of AMPs. This paper describes the purification and characterization of the novel
Fish live in a microbe-rich environment thus requiring them to antimicrobial peptide, pelteobagrin, from the skin mucus of yellow
have a robust system of host defense. Fish skin is at particularly high catfish. The present study also demonstrates the immune defensive
risk of infection since it is constantly exposed to water-borne role of epidermal mucus in fish innate immune system.
microorganisms. Yet, compared to that of mammals and amphibians,
the adaptive immune system of fish is relatively under developed 2. Materials and methods
(Pasquier, 2001). Thus, fish are thought to have a great reliance on
their innate immune system. The skin mucus provides fish with the 2.1. Animals and mucus collection

The yellow catfish of both sexes (n = 8, mass range of 120–170 g)

⁎ Tel.: +86 25 83065370; fax: +86 25 86188987. were purchased from local market in Nanjing, China. Mucus was
E-mail address: suyueju@[Link]. collected by gently scraping the dorsal body of the fish with a plastic

1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/[Link].2010.11.002
150 Y. Su / Comparative Biochemistry and Physiology, Part B 158 (2011) 149–154

spatula. Ventral skin mucus was not collected to avoid intestinal and Bacillus subtilis (ATCC6633) and Candida albicans (ATCC2002). The
sperm contamination. The skin mucus harvest was immediately minimal inhibitory concentration (MIC) of pelteobagrin against these
homogenized in 50 mM phosphate buffer solution (PBS), pH 6.0 microorganisms was determined by microdilution method (Belén
containing 5 mM EDTA on ice by using a glass homogenizer. Insoluble et al., 2007). Briefly, the tested bacteria were first grown in Luria–
substances were removed by centrifuging at 15,000 g for 30 min, and Bertani broth (Invitrogen, Carlsbad, CA, USA) until the desired density
the supernatant was collected for further purification. was reached (OD600 = 0.6). For E. coli and S. aureus, the bacteria
suspensions were then adjusted to 2 × 105 bacteria/mL in LB broth and
2.2. Peptide purification used for subsequent assay. The synthesized peptide was dissolved in
sterile distilled water and two-fold diluted. The assay mixture
The supernatant (20 mL) was filtered through a 5 kDa cut-off contained 10 μL two-fold diluted peptide sample or water, 40 μL
Centriprep filter (Millipore, Bedford, CA) to remove high molecular bacterial suspension and 50 μL LB broth. To investigate the effect of
mass proteins. After that, the filtrate (total 8 mL, each batch of 200 μL) salt on antimicrobial activity of pelteobagrin, a final concentration of
was applied to a 5 mm×300 mm hypersil ODS25 μm reversed-phase NaCl ranging from 0 to 548 mM was added to the assay mixture. All
high-performance liquid chromatography (RP-HPLC) column equilibrat- the assays were carried out in a 96-well sterile polypropylene
ed with 0.1% (v/v) trifluoroacetic acid (TFA)/water. Elution (0.8 mL/min) microtiter plate (Costar, Corning, NJ, USA) with final peptide
was performed using 0.1% (v/v) TFA/water over 5 min, followed by a concentrations ranging from 256 to 0.25 μg/mL. After 16–18 h of
linear gradient of 0–80% acetonitrile containing 0.1% (v/v) TFA over incubation at 37 °C, the inhibition of bacterial growth was assessed by
75 min, and final elution with 100% acetonitrile containing 0.1% (v/v) TFA. lack of cloudiness in the well, and MIC was defined as the lowest
The eluate was monitored at 215 nm, and UV-absorbing peaks were concentration of peptide that inhibited visible cell growth. For C.
collected, concentrated to a small volume by vacuum centrifugation, and albicans, incubation was carried out in RPMI 1640 medium for 48 h at
assayed for antimicrobial activity. The fractions with antimicrobial 35 °C. All assays were performed twice with duplication.
activity were pooled and further purified by C18 RP-HPLC under modified
conditions (Fig. 1A, B) and concentrated as before.
2.7. Hemolysis assays
2.3. Peptide sequencing and mass spectrometry determination
Rabbit red blood cells were used to test the hemolytic ability of
Complete peptide sequencing was undertaken by Edman degrada- pelteobagrin as reported (Bignami, 1993). The synthesized peptide
tion on an Applied Biosystems pulsed liquid-phase sequencer, model was serially diluted and incubated with rabbit red blood cells in
492cLC (Applied Biosystems, Foster City, CA, USA). Mass measurement phosphate buffered saline at 37 °C for 30 min. After centrifugation, the
was performed on an Applied Biosystems (ABI) Voyager DR-STE absorbance of the supernatant was measured at 540 nm. Maximum
instrument using α-cyano-4-hydroxycinnamic acid as the ionization hemolysis was determined by adding 1% Triton X-100 to a sample of
matrix. The peptide was dissolved in 0.1% TFA in water (1 μg/μL) and cells.
diluted with 3 vol. of 50 mM α-cyano-4-hydroxycinnamic acid in 80%
acetonitrile.
2.8. Transmission electron microscopy
2.4. Primary structure analysis and characterization
Transmission electron microcopy was utilized to study the potential
Homology searches of the isolated antimicrobial peptide were mechanism of pelteobagrin acting on Gram-positive bacteria S. aureus,
performed on the SwissProt database using the basic local alignment using the methods described by Friedrich et al. (2000) with minor
search tool (BLAST) (Altschul et al., 1997) provided by the National modifications. Briefly, exponential-phase bacteria were treated with the
Center for Biotechnology Information (NCBI) Server ([Link] peptide of 10 × MIC for 30 min at 37 °C, and then centrifuged at 300 g for
[Link]/BLAST) and peptide databases ([Link] 10 min. The bacterial pellets were fixed with 2.5% buffered glutaralde-
edu/AP/database/[Link]). The charge and molecular mass were hyde for 1 h. The cells were then post-fixed with 1% buffered osmium
predicted using the Expert Protein Analysis System (ExPASy) tetroxide for 1 h, stained en bloc with 1% uranyl acetate, dehydrated in a
proteomics tool at the Swiss Institute of Bioinformatics (http:// graded series of ethanol, and finally embedded in white resin. Thin
[Link]/). The Shiffer–Edmundson helical wheel diagram was sections (LKB-V, Sweden) on copper grids were stained with 1% uranyl
created by using the Protean subprogram of the DNASTAR software. acetate and lead citrate. The buffer used was 0.1 M sodium cacodylate,
pH 7.4. The resin and grids were purchased from Marivac (Halifax, NS,
2.5. Peptide synthesis Canada). Imaging was performed with a HITACH-H 7500 microscope
under standard operating conditions.
Pelteobagrin was synthesized by solid phase synthesis on an
Applied Biosystems model 433A peptide synthesizer according to the
manufacturer's standard protocols. After cleavage, the crude synthetic 3. Results
peptide was purified on a Vydac 218TP510 C18 reverse phase-HPLC
column (25 × 1 cm) eluted at a flow rate of 2 mL/min by a linear 3.1. Peptides purification
gradient of acetonitrile in 0.1% trifluoroacetic acid in water. The purity
and identity of the peptide were confirmed by automated Edman Since most AMPs are smaller than 5 kDa, the supernatant of the
sequencing and mass spectrometry analysis. The synthetic peptide yellow catfish skin mucus was first filtered through a 5 kDa cut-off
was then evaluated for biological activity. The peptide was quantified Centriprep filter (Millipore, Bedford, CA, USA) to remove high
by UV absorbance at 215 and 225 nm using the formula: concentra- molecular mass proteins, then run on a 5 mm × 300 mm hypersil
tion (mg/mL) = (A215–A225) × 0.144. ODS25 μm reversed-phase high-performance liquid chromatography
(RP-HPLC) column. Antimicrobial activity was assessed for each peak
2.6. Antimicrobial assay and the active fraction (Fig. 1A) was collected and further purified by
C18 RP-HPLC with a shallow gradient. This resulted in one main
Antimicrobial activity was tested against the following bacteria: fraction containing antibacterial activity (Fig. 1B) corresponding to a
Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC25922), peak with retention time of 19.3 to 20.3 min.
 Y. Su / Comparative Biochemistry and Physiology, Part B 158 (2011) 149–154 151

3.2. Structure and sequence determination spectrometry was 2244.4 Da, which matched well with the theoretical
molecular mass (2244.8) (Fig. 1C). When searched in the SwissProt,
The purified peptide was completely sequenced by Edman degra- NCBI, and peptide databases, no significant similarities were found
dation methods, and the following sequence GKLNLFLSRLEILKLFVGAL between it and any other bioactive peptides, suggesting this peptide was
was obtained. The molecular mass analyzed by MALDI-TOF mass novel. Thus, the peptide was assigned the name pelteobagrin. An

Fig. 1. Chromatography of purifying pelteobagrin from the skin mucus of yellow catfish. The filtrate was loaded on a 5 mm × 300 mm hypersil ODS25 μm reversed-phase high-
performance liquid chromatography (RP-HPLC) column equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA)/water. Elution was carried out with the indicated gradient of
acetonitrile in (A) at a flow rate of 0.8 mL/min, and fractions were tested for antimicrobial activities. The fraction with antimicrobial activity indicated by an arrow in (A) was further
purified by RP-HPLC under the modified conditions indicated in (B) at a flow rate of 0.8 mL/min. The purified antimicrobial peptide was indicated by an arrow in (B). Dotted line
indicates the acetonitrile concentration. The purified peptide was analyzed by MALDI-TOF mass spectrometry (C).
152 Y. Su / Comparative Biochemistry and Physiology, Part B 158 (2011) 149–154

alignment of pelteobagrin with several fish skin-derived AMPs is shown

in Fig. 2. Pelteobagrin is predicted to have a positive charge of +2
(pI = 9.99) and has about 60% hydrophobic amino acid content.
Schiffer–Edmundson helical wheel modeling was employed to predict
secondary structure of pelteobagrin (Fig. 3). The diagram indicated
that ten out of twelve hydrophobic residues are on the same surface,
while four out of six hydrophilic residues are located on the opposing
side, implying the possibility that pelteobagrin forms an amphipathic
alpha-helix.

3.3. Antimicrobial activity and salt sensitivity

The antimicrobial activity of natural and synthetic pelteobagrin was

examined against four microorganisms (Table 1). The natural and
synthetic pelteobagrin were equally active against all tested micro-
organisms. Notably, B. subtilis was most sensitive, with a MIC of 2 μg/mL.
Pelteobagrin was determined to be lethal to the sensitive microorgan-
isms since they were not capable of resuming growth on agar plates
after 6 h treatment with concentrations above the corresponding MICs.
Low concentrations of NaCl (0–137 mM) had no effect on the
antimicrobial activity of pelteobagrin; while 274 mM NaCl almost Fig. 3. Schiffer–Edmundson helical wheel diagram demonstrating probable amphi-
completely abolished its antimicrobial activity. Despite being still active pathic α-helical conformation of pelteobagrin. The black circles indicate hydrophobic
amino acids; the white circles indicate hydrophilic amino acids. Glycine is considered a
against B. subtilis, the MIC value increased 64-fold (Table 1).
neutral residue and is shown with both black and white circles. Residue numbers
counting from amino terminal are shown.
3.4. Hemolytic activity
important for them to insert and form ion channel in the bacteria
When pelteobagrin was incubated with rabbit red blood cells at a
membrane, further induce strong permeability changes and subse-
concentration of 1–400 μg/mL, no change in absorbance (540 nm) was
quently causes cell lysis (Park et al., 1998a; Campagna et al., 2007). In
observed. There was no detectable hemolytic activity for pelteobagrin at
this study, a novel antimicrobial peptide, named pelteobagrin, was
concentrations 12-fold higher than its antimicrobial activity.
isolated from the skin mucus of yellow catfish. Pelteobagrin consists
of 20 amino acids with a net charge +2. Although it is not perfect, the
3.5. Transmission electron microscopic analysis secondary structural analysis shows that pelteobagrin might share
this common feature of other AMPs. However, further investigation
To investigate the possible mechanism of pelteobagrin acting on should be done to determine it. Antimicrobial assays showed that
microorganism, the Gram-positive bacterium S. aureus was examined pelteobagrin was active against Gram-positive and Gram-negative
via TEM. As shown in Fig. 4, after treatment with pelteobagrin, the cell bacteria as well as a fungus. All these suggest that pelteobagrin might
wall appeared turgid, and laminar mesosomes arose from the septa be a key component of the innate defense system in yellow catfish.
and cell wall as indicated by the arrows, while the untreated bacteria Previous studies on fish skin-derived AMPs indicate that their
had no such structures in them. antimicrobial activity is correlated with the habitat of the fish species.
The AMPs isolated from seawater fish species such as pleurocidin, a
4. Discussion histone H2B-derived peptide of cod epidermal mucus, and Myxinidin
are salt-tolerant (Cole et al., 1997; Bergsson et al., 2005; Subramanian
Today, it has been recognized that fish skin mucus plays an important et al., 2009), whereas oncorhyncin III isolated from the epidermal
role in the host defense system because it contains a collection of mucus of anadromous rainbow trouts showed poor activity at higher
antibacterial substances, including AMPs (Subramanian et al., 2008). salt concentrations (Fernandes et al., 2003). Since yellow catfish is
During the past few years, a few AMPs have been identified from fish restricted to freshwater habitats, pelteobagrin is speculated to
epidermal mucus including pardaxin, pleurocidin, parasin, oncorhyncin perform its function only at low concentrations of NaCl, and the salt
III, oncorhyncin II, SAMP-H1, a histone H2B derived peptide from Atlantic sensitive assay further corroborated this.
cod, and Myxinidin (Oren and Shai, 1996; Cole et al., 1997; Park et al.,
1998b; Birkemo et al., 2003; Fernandes et al., 2003; Fernandes et al.,
2004; Lüders et al., 2005; Bergsson et al., 2005; Subramanian et al., Table 1
2009). Antimicrobial activity of pelteobagrin under different concentrations of NaCl.
One common feature of many AMPs is that they possess an
MICa (μg/mL)
amphipathic alpha-helix structure, and this structure is probably
Microorganisms

NaCl (mM) E. coli S. aureus B. subtilis C. albicans

Pelteobagrin
0 16 4 2 64
Myxinidin 69 16 4 2 64
137 16 4 2 64
Pleurocidin 274 N 256 N 256 128 N256
548 N 256 N 256 N256 N256
Pardaxin-P1
These concentrations represent mean values of two independent experiments
Fig. 2. Comparison of the amino acid sequences of pelteobagrin, Myxinidin performed in duplicates.
(Subramanian et al., 2009), pleurocidin (Cole et al., 1997), and pardaxin-P1 (Oren N, The highest concentration tested caused no inhibition.
a
and Shai, 1996). As aligned by the ClustalW2 program. Gaps have been introduced to Minimal peptide concentration required for total inhibition of cell growth in liquid
maximize sequence similarities. Identical amino acids are in bold. medium.
 Y. Su / Comparative Biochemistry and Physiology, Part B 158 (2011) 149–154 153

Fig. 4. Ultrastructure of S. aureus treated by pelteobagrin (40 μg/mL). (A) Control; and (B) treated by pelteobagrin. Mesosome structures are indicated by arrows. CW: cell wall.

The cytotoxicity of many antimicrobial peptides to mammalian properties of pelteobagrin suggest that it may have utility in
cells greatly limits their use as therapeutics. When tested on rabbit red aquaculture and human health-related applications.
blood cells, pelteobagrin showed no hemolytic activity even at
peptide concentrations of up to 12-fold higher than its MIC. This Acknowledgements
indicates that pelteobagrin may have potential uses in human health-
related applications. I would like to thank Neil W. Ross (National Research Council of
Mesosomes, which are intracytoplasmic membrane inclusions, Canada) and Andrew T. Gewirtz (Emory University) for critical reading
have been regarded as structural artifacts induced by the chemical of this paper.
fixatives used on the cells prior to plastic embedding and thin
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