A novel antimicrobial peptide from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen).

Abstract

A novel lumbricin-like antimicrobial peptide named lumbricin-PG was isolated from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen), using a procedure of one step Sephadex G-50 gel filtration and one step C(8) reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as FSRYARMRDSRPWSDRKNNYSGPQFTYPPEKAPPEKLIKWNN EGSPIFEMPAEGGHIEP by Edman degradation combined with cDNA cloning and mass spectrometry analysis. The cDNA encoding lumbricin-PG was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 73 amino acid residues including a mature lumbricin-PG and predicted signal peptide. It showed similarity with lumbricin antimicrobial peptide from the earthworm, Lumbricus rubellus by BLAST search. Purified lumbricin-PG exerted potential antimicrobial activities against bacteria and fungi; it showed weak hemolysis activity against human and rabbit red cells.

Full text

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Peptides 32 (2011) 1146–1150
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
A novel antimicrobial peptide from skin secretions of the earthworm,
Pheretima guillelmi (Michaelsen)
Wenliang Lia,b,1, Sisi Lib,1, Jian Zhongc,d,1, Zhu Zhua, Jingze Liub,∗, Wenhong Wangc,∗∗
aOncology Department, The First Affiliated Hospital of Kunming Medical College, Kunming 650032, Yunnan, China
bKey Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050016, China
cThe Affiliated Zhongda Hospital of Southeast University, Nanjing 21009, Jiangsu, China
dNanjing Maternal and Child Health Hospital, 123# Tianfeixiang, Baixia District, Nanjing 210004, Jiangsu, China
a r t i c l e i n f o
Article history:
Received 27 March 2011
Received in revised form 14 April 2011
Accepted 14 April 2011
Available online 22 April 2011
Keywords:
Earthworm
Antimicrobial peptide
Skin secretion
Lumbricin
Pheretima guillelmi
a b s t r a c t
A novel lumbricin-like antimicrobial peptide named lumbricin-PG was isolated from skin secretions of
the earthworm, Pheretima guillelmi (Michaelsen), using a procedure of one step Sephadex G-50 gel fil-
tration and one step C8reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino
acid sequence was determined as FSRYARMRDSRPWSDRKNNYSGPQFTYPPEKAPPEKLIKWNN EGSPIFEM-
PAEGGHIEP by Edman degradation combined with cDNA cloning and mass spectrometry analysis. The
cDNAencodinglumbricin-PGwasclonedbycDNAlibraryscreening.ThepredictedproteinfromthecDNA
sequence was composed of 73 amino acid residues including a mature lumbricin-PG and predicted signal
peptide.Itshowedsimilaritywithlumbricinantimicrobialpeptidefromtheearthworm,Lumbricusrubel-
lus by BLAST search. Purified lumbricin-PG exerted potential antimicrobial activities against bacteria and
fungi; it showed weak hemolysis activity against human and rabbit red cells.
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
Antimicrobial peptides have become considered as important
and ubiquitous components in innate immunity to resist microor-
ganism infection [3,9,20,22,25]. More than 1000 antimicrobial
peptides have been found in both invertebrates and vertebrates,
with many still under discovering [9,11,12,23]. Most of inverte-
brate antimicrobial peptides are from arthropods, such as insects
and ticks [2,9,25]. In vertebrates, many antimicrobial peptides
are identified from amphibians [5,6,8,10–12,14–16,19,24–28]. A
wide variety of structural motifs have been in antimicrobial pep-
tides. Based on their structural motifs, antimicrobial peptides
can divided into several groups including linear peptides forming
amphipathic and hydrophobic helices, cyclic peptides and small
proteins forming ?-sheet structures, peptides with unique amino
acid compositions, cyclic peptides with thio-ether groups in the
ring, lipopeptides terminating in an amino alcohol and macrocyclic
knotted peptides [9–12].
Earthworms are ancient creatures, which have existed on the
earth at least 700 million years [4]. Most of their lives are in soils,
which are likely laden with pathogens. Earthworms are forced to
∗Corresponding author. Tel.: +86 311 86268313; fax: +86 311 86268313.
∗∗Corresponding author.
E-mail addresses: jzliu21@heinfo.net (J. Liu), wangwenhong@hotmail.com
(W. Wang).
1These authors share the same contribution to this paper.
adopt and survive in this kind of environments [17]. Their naked
skins play key roles in the everyday survival of amphibians and
their ability to exploit a wide range of habitats and ecological
conditions, and act as the first line to against noxious aggres-
sion by microorganisms, parasites, and predators. Therefore, they
likely contain an excellent chemical defense system composed of
pharmacological and antimicrobial peptides. Several anti-infection
components including lysozymes, fetidins, and antimicrobial pep-
tideshavebeenfoundinearthworms[4,13,21].Althoughmorethan
3000 species of earthworms are known, only three antimicrobial
peptides are identified from three species [4,13,21]. In this study,
we report the purification, characterization and cDNA cloning of a
antimicrobialpeptidefromtheearthwormskinsecretionsofPhere-
tima guillelmi.
2. Materials and methods
2.1. Earthworms
Earthworms of mature P. guillelmi (weight, 0.8–1g, 15–20cm
length; N=1000) are collected from Jiangsu province of China.
2.2. Collection of skin secretions
Earthworms were washed by 0.1M phosphate buffer solu-
tion (PBS), pH 6.0, containing protease inhibitor cocktail (Sigma,
P2714). The collected solutions (500ml of total volume) were
0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2011.04.015

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W. Li et al. / Peptides 32 (2011) 1146–1150
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Fig. 1. Purification of antimicrobial peptide from skin secretions of P. guillelmi.
(A) Sephadex G-50 gel filtration of skin secretions of P. guillelmi. The skin secre-
tions were applied on a Sephadex G-50 column equilibrated with 0.1M phosphate
buffer, pH 6.0. Elution was performed with the same buffer, collecting fractions of
3.0ml. (B) The peak (indicated as III) with antimicrobial activity from Sephadex G-
50 were further purified on a Hypersil BDS C8RP-HPLC column equilibrated with
0.1% (v/v) trifluoroacetic acid in water. The elution was performed with the gradi-
ents of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water shown in (B) at a flow
rate of 0.7ml/min, and fractions were tested for antimicrobial activity. The purified
antimicrobial peptide is indicated as an arrow.
quickly centrifuged at 5000×g for 20min and the supernatants
were lyophilized. The crude sample (15g) was stored at −20◦C.
2.3. Antimicrobial peptide purification
The aliquot (1g) of lyophilized crude sample was dissolved in
10ml 0.1M PBS, pH 6.0, containing the protease inhibitor cock-
tail and centrifuged at 5000×g for 10min. The supernatant was
applied to a Sephadex G-50 (Superfine, Amersham Biosciences,
2.6×100cm)gelfiltrationcolumnequilibratedwith0.1MPBS.Elu-
tion was performed with the same buffer, collecting fractions of
3.0ml. The absorbance of the elute was monitored at 280nm. The
antimicrobial assay was determined as described below. The frac-
tions containing antimicrobial activities were pooled, lyophilized,
and re-suspended in 0.1M PBS, pH 6.0, and purified further by
C8reverse-phase high-performance liquid chromatography (RP-
HPLC, Hypersil BDS C8, 30cm×0.46cm) using WatersTM600
system as illustrated in Fig. 1.
2.4. Structural analysis
The eluted fractions containing antimicrobial activity from
RP-HPLC were analyzed by a matrix-assisted laser desorption ion-
ization time-of-flight mass spectrometer (MALDI-TOF-MS) AXIMA
CFR (Kratos Analytical) in positive ion and linear mode. The opera-
tion parameters were as follows: the ion acceleration voltage was
20kV, the accumulating time of single scanning was 50s, polypep-
Fig. 2. Purified antimicrobial peptide (lumbricin PG) was digested by sequencing
grade modified trypsin (Promega) in 0.05M NH4HCO3 (pH 7.8) at 37◦C for 12h
and tryptic peptides were isolated on a C18 RP-HPLC column (Hypersil BDS C18,
25cm×0.46cm) using WatersTM600 system. Recovered peptide fragments (indi-
cated by arrows) were subjected to Edman degradation analysis.
tidemassstandard(KratosAnalytical)servingasexternalstandard.
The accuracy of mass determinations was within 0.1%. Purified
peptide (0.75mg) was incubated with 0.01mg sequencing grade
modified trypsin (Promega) in 0.05M NH4HCO3(pH 7.8) at 37◦C
for 12h. Tryptic peptides were isolated on a C18RP-HPLC column
(Hypersil BDS C18, 25cm×0.46cm) using WatersTM600 system at
30◦C and at a flow rate of 0.1ml/min. The solvent system used was
0.1%trifluoroaceticacidinH2O(solventA)and0.09%trifluoroacetic
acid in 100% acetonitrile (solvent B) (Fig. 2). Amino acid sequences
of the N-terminus and interior peptide fragments recovered from
trypsin hydrolysis were determined by automated Edman degra-
dation on an Applied Biosystems pulsed liquid-phase sequencer,
model 491.
2.5. Construction and screening of a cDNA library
SMARTTMtechnique was used to synthesize cDNA by using a
SMARTTMPCR cDNA synthesis kit (Clontech, Palo Alto, CA). mRNA
waspreparedbyoligo(dT)cellulosechromatographyusingthetotal
RNA extracted from P. guillelmi skins by TRIzol (Life Technologies
Ltd.). A directional cDNA library was constructed with a plasmid
cloning kit (SuperScriptTMPlasmid System, GIBCO/BRL) following
the instructions of the manufacturer, producing a library of about
1.2×106independent colonies.
A PCR-based method for high stringency screening of DNA
libraries was used for screening and isolating the clones with
somemodifications. Twooligonucleotide
tt(t/c)tc(a/t/g/c)cg(a/t/c/g)ta(t/c)gc(a/t/c/g)cg(a/t/c/g)atgcg-3?,
in the sense direction), a specific primer designed according to
the amino acid sequence determined by Edman degradation and a
vector SP6promoter primer (5?-CATACGATTTAGGTGACACTATAG-
3?, in the antisense direction) located in the 3?part of the cloned
insert, were used in PCR reactions. All the oligonucleotide primers
for PCR were prepared with a DNA synthesizer (Model 381A,
Applied Biosystems). The PCR conditions were: 2min at 94◦C,
followed by 30 cycles of 10s at 92◦C, 30s at 52◦C, 60s at 72◦C.
DNA sequencing was performed on an Applied Biosystems DNA
sequencer, model ABI PRISM 377.
primers,S1,5?-
2.6. Antimicrobial assays
Standard
crobial
aureus (ATCC2592), Gram-negative bacterium Escherichia coli
(ATCC25922) and Pseudomonas aeruginosa (ATCC27853), and
fungus Candida albicans (ATCC2002) were obtained from Nanjing
Medical University. The antimicrobial assay was according to the
bacterial
assays,
and fungalstrains
bacterium
used in antimi-
Gram-positive
Staphylococcus

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W. Li et al. / Peptides 32 (2011) 1146–1150
Fig. 3. cDNA encoding lumbricin PG from the skin secretions of P. guillelmi and deduced amino acid sequence. The amino acid sequences of peptide fragments determined
by Edman degradation are underlined. The predicted signal peptide is italic. (–) Stop codon.
method described by Li et al. [11,12]. Bacteria were first cultured
in LB (Luria–Bertani) broth. After their growth up to an OD600nmof
0.8, a 10?l aliquot of the bacteria was then taken and added to 8ml
of fresh LB broth with 0.7% agar and poured over a 90mm Petri
dish containing 25ml of 1.5% agar in LB broth. After the top agar
hardened, a 20?l aliquot of the test sample filtered on a 0.22?m
Millipore filter was dropped onto the surface of the top agar and
completely dried before being incubated overnight at 37◦C. If an
examined sample contained antimicrobial activity, a clear zone
formed on the surface of the top agar representing inhibition of
bacterial growth. Minimal inhibitory concentration (MIC) was
determined in liquid LB medium as previous report [11]. The MIC
at which no visible growth occurred was recorded.
2.7. Hemolysis assays
Hemolysisactivitieswereevaluatedusinghumanandrabbitred
cellsinAlsever’ssolution(ing/L:NaCl,4.2;citricacid3Na2H2O,8.0;
citric acid H2O, 0.55; d-glucose, 20.5) as reported [2]. The tested
samples were serially diluted by Alsever’s solution and incubated
with red cells at 37◦C for 30min. The cells were centrifuged and
the absorbance in the supernatant was measured at 595nm. 100%
hemolysis was determined by adding 1% Triton X-100 to a sample
of cells.
2.8. Synthetic peptides
AllpeptidesusedforbioassaysweresynthesizedbyGLBiochem
(Shanghai) Ltd. (Shanghai, China) and analyzed by HPLC and mass
spectrometry to confirm purity higher than 98%.
3. Results
3.1. Purification of antimicrobial peptide
The skin secretion sample of P. guillelmi was fractionated into
fivefractionsbySephadexG-50gelfiltrationasillustratedinFig.1A.
Antimicrobial activities were found to be concentrated on fractions
III. Fractions III was subjected to further purification by the C8RP-
HPLC using indicated acetonitrile gradient in Fig. 1B. More than
30 peaks were eluted by the C8RP-HPLC as illustrated in Fig. 1. A
peak (indicated by an arrow in Fig. 1B), which was found to contain
antimicrobial activities, was eluted out at the acetonitrile concen-
trationof65%.ThispeakwerecollectedandanalyzedbyMALDI-TOF
mass spectrometry and Edman degradation.
3.2. Amino sequencing and molecular weight
Purifiedantimicrobialpeptide(total2mg)indicatedbyanarrow
in Fig. 1 was named lumbricin-PG. MALDI-TOF-MS analysis gave an
observed mass of 6909.07. Its amino acid sequences of N-terminus
and interior peptide fragments were determined by automated
Edman degradation as illustrated in Fig. 3.
3.3. cDNA cloning
Aclonecontaininganinsertaround529-basepairswasscreened
andisolated.Bothstrandsofthecloneweresequenced(Fig.3).This
cDNA was found to have an open reading frame that encodes a
polypeptide precursor composed of 73 amino acid residues includ-
ing the mature lumbricin-PG sequence. The amino acid sequence
deduced from the cDNA sequence is identical to the amino acid
sequencedeterminedbyEdmandegradation.Thepredictedmature
lumbricin-PGcontains59aminoacidresiduesincluding9basicand
8acidicaminoacidresiduesandnohalf-cysteine.Lumbricin-PGhas
a theoretical molecular mass of 6908.7, which matched well with
the observed molecular mass of 6909.07 from mass spectrometry
analysis. By BLAST search, it shared obvious similarity with lum-
bricin antimicrobial peptide from the earthworm, L. rubellus [4]
(Fig. 4).
3.4. Antimicrobial activity
Purified lumbricin-PG exhibited potential antimicrobial activity
against the tested strains as shown in Table 1. Among the tested
strains, P. aeruginosa and S. aureus were the most sensitive to this
antimicrobial peptide. The MIC is 2.5 and 5.0?g/ml, respectively.
It had the weakest antimicrobial activity (MIC of 20?g/ml) against
E. coli. The sensitive strains were not capable of resuming growth
on agar plates after a 6-h treatment with concentrations above the
corresponding MICs.
3.5. Hemolytic activity
Some antimicrobial peptides were found to destroy membrane
and exhibited hemolytic activities [10,11]. Human and rabbit red
blood cells were used to examine lumbricin-PG’s hemolytic capa-

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W. Li et al. / Peptides 32 (2011) 1146–1150
1149
Fig. 4. Sequence alignment of antimicrobial peptides from earthworms. Lumbricin PG is from this report; lumbricin I is from Ref. [4]; PP-1 is from Ref. [21]; *identical amino
acid residue; hyphen: the sign was introduced for optimal alignment.
bility in current experiments. As a result, lumbricin-PG had little
hemolytic activity on both human and rabbit red blood cells. At the
concentration of 200?g/ml, it induced 1.5 and 2.1% hemolysis of
human and rabbit red blood cells, respectively.
4. Discussion
Themajorityoftheantibioticsusedtodayoriginatefromnatural
products at the “golden age” of antibiotics discovery (the 1940s to
the1970s).Mostnaturalproduct-basedcandidatescurrentlyunder
development are modified, improved versions of old drugs [7].
Because of the development of antibiotic resistance in virtually all
clinicallyimportantpathogens,alternativestoconventionalantibi-
otics, which have new type of antimicrobial mechanism or modes,
areurgentlyneeded.Althoughtheactualantimicrobialmechanism
remains still unclear, as a novel class of antibiotics, antimicro-
bial peptides have attracted considerable attentions, especially for
thetreatmentofantibiotic-resistantpathogens.Antimicrobialpep-
tides have been considered to contain low potential to induce
drug-resistance of microorganisms [5,9,25]. As promising candi-
dates to treat pathogens with drug-resistance against traditional
antibiotics, antimicrobial peptides are attracting much attention to
study and developing as new type of clinical anti-infective agents.
In addition, much work is also performed to identify novel antimi-
crobial peptides.
Earthworm species have rich diversity. Based on their living
environments, it is rational to think that there are effective anti-
infective agents in earthworm’s skins. Cho et al. identified the first
antimicrobial peptide (lumbricin I) from the earthworm, L. rubel-
lus [4]. Lumbricin I is considered as a proline-rich antimicrobial
peptide containing 62 amino acids including proline (15%) with
a molecular weight of 7231Da. Lumbricin I showed antimicro-
bial activity in vitro against a broad spectrum of microorganisms
withouthemolyticactivity[4].Recently,twoanotherantimicrobial
peptides(PP1andOEP3121)havebeenidentifiedfromearthworms
of Pheretima tschiliensis and Eisenia foetida, respectively [13,21].
PP1 shows 77.6% sequence similarity with lumbricin I but it lacks
an obvious signal peptide sequence (Fig. 4). Surprisingly, OEP3121
only has 5 amino acid residues (ACSAG) [13].
The antimicrobial peptide, lumbricin-PG was identified from
skin secretions of the earthworm, P. guillelmi in the current work.
Maturelumbricin-PGiscomposedof59aminoacidresiduesinclud-
Table 1
Antimicrobial activity of lumbricin-PG.
MicrooganismMIC (?g/ml)
Lumbricin PG
Gram-positive bacteria
S. aureus
Gram-negative bacteria
E. coli
P. aeruginosa
Fungus
C. albicans
5.0
20.0
2.5
10.0
MIC, minimal peptide concentration required for total inhibition of cell growth in
liquid medium. These concentrations represent mean values of three independent
experiments performed in duplicates.
ing 9 prolines. It shares 66% identity with lumbricin I from L.
rubellus. Based on their high sequence identity, lumbricin-PG is
likely a homologue of lumbricin I. This result implies that differ-
ent genus of earthworms share the same family of antimicrobial
peptides. Although multiple prolines are found in sequences of
lumbricins, the propotion (15%) is much less than that of proline-
rich antimicrobial peptides, such as some cathelicidins [1]. In
addition, multiple aromatic amino residues including Phe, Tyr,
or Trp (12–14%) are in the sequences of lumbricins. It has been
reported that aromatic (Phe or Trp) residue addition can increase
broadspectrumactivityofantimicrobialpeptide[18].Multiplearo-
matic residues in lumbricins may contribute their broad spectrum
antimicrobial activities. Multiple aromatic residues and prolines
might be a significant feature in the antimicrobial peptide family
of lumbricin.
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