Báo cáo khoa học: Novel cathelicidin-derived antimicrobial peptides from Equus asinus docx

Novel cathelicidin-derived antimicrobial peptides from
Equus asinus
Zekuan Lu
1
*, Yipeng Wang
2,3
*, Lei Zhai
1
, Qiaolin Che
3
, Hui Wang
1
, Shuyuan Du
1
, Duo Wang
1
,
Feifei Feng
1,2
, Jingze Liu
1
, Ren Lai
3
and Haining Yu
1,2
1 College of Life Sciences, Hebei Normal University, Shijiazhuang, China
2 School of Life Science and Biotechnology, Dalian University of Technology, China
3 Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming, Yunnan, China
Introduction
Cathelicidins are a family of structurally diverse anti-

microbial peptides found in virtually all species of
mammals that play a critical role in the innate immune
system [1,2]. They are characterized by a N-terminal
signal peptide (30 residues) and a highly conserved
cathelin domain (99–114 residues long) followed by a
C-terminal mature peptide (12–100 residues) that is
characterized by a remarkable structural variety [3].
Cathelicidins are most abundantly present in circulat-
ing neutrophils and myeloid bone marrow cells [4],
Keywords
cathelicidin; Equus asinus; function; gene
cloning; peptide identification
Correspondence
R. Lai, Key Laboratory of Animal Models
and Human Disease Mechanisms, Kunming
Institute of Zoology, Chinese Academy of
Sciences, Kunming 650223, Yunnan, China
Fax ⁄ Tel: +86 871 5196202
E-mail:
H. Yu, College of Life Sciences, Hebei
Normal University, Shijiazhuang, Hebei
050016, China
Fax ⁄ Tel: +86 311 86268842
E-mail:
*These authors contributed equally to this
work
(Received 11 January 2010, revised 10
March 2010, accepted 15 March 2010)
doi:10.1111/j.1742-4658.2010.07648.x
In the present study, EA-CATH1 and EA-CATH2 were identified from

a constructed lung cDNA library of donkey (Equus asinus) as members
of cathelicidin-derived antimicrobial peptides, using a nested PCR-based
cloning strategy. Composed of 25 and 26 residues, respectively,
EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins
and have no sequence homology to other cathelicidins identified to date.
Chemically synthesized EA-CATH1 exerted potent antimicrobial activity
against most of the 32 strains of bacteria and fungi tested, especially
the clinically isolated drug-resistant strains, and minimal inhibitory con-
centration values against Gram-positive bacteria were mostly in the
range of 0.3–2.4 lgÆmL
)1
. EA-CATH1 showed an extraordinary serum
stability and no haemolytic activity against human erythrocytes in a
dose up to 20 lgÆmL
)1
. CD spectra showed that EA-CATH1 mainly
adopts an a-helical conformation in a 50% trifluoroethanol ⁄ water solu-
tion, but a random coil in aqueous solution. Scanning electron micro-
scope observations of Staphylococcus aureus (ATCC2592) treated with
EA-CATH1 demonstrated that EA-CATH could cause rapid disruption
of the bacterial membrane, and in turn lead to cell lysis. This might
explain the much faster killing kinetics of EA-CATH1 than conventional
antibiotics revealed by killing kinetics data. In the presence of CaCl
2
,
EA-CATH1 exerted haemagglutination activity, which might potentiate
an inhibition against the bacterial polyprotein interaction with the host
erythrocyte surface, thereby possibly restricting bacterial colonization and
spread.
Abbreviations

cfu, colony-forming units; MH, Mueller–Hinton broth; MIC, minimal inhibitory concentration; SEM, scanning electron microscope.
FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2329
and are also found in mucosal epithelial cells and skin
keratinocytes [5].
To date, a number of cathelicidins have been identi-
fied from mammals, such as humans, monkeys, mice,
rats, rabbits, guinea pigs, pigs, cattle, sheep, goats and
horses [6–8]. According to secondary structures, these
cathelicidins are further divided into three groups [6].
Group one possesses an amphipathic a-helical struc-
ture (human, mouse and bovine BMAP-34 peptides).
Group two, including porcine PR-39 and bovine bacte-
necins, is characterized by a high content of one or
two amino acids, often proline and arginine. The third
group mainly adopts a b-sheet structure, such as in
protegrins.
Apart from the primary antimicrobial activities,
certain cathelicidins also participate in wound repair,
the induction of angiogenesis and cytolysis, chemo-
taxis for neutrophils, monocytes, mast cells and T
cells [6,9]. Human cathelicidin LL-37 was reported to
have antitumour and anti-HIV activities [10]. Con-
cordant with these important roles in host defence
and disease resistance, the aberrant expression of
cathelicidins is often associated with various disease
processes [11]. Therefore, future studies on the
biological activities and clinical purposes of cathelici-
dins will undoubtedly facilitate the treatment of
infectious diseases, in addition to offering more novel
therapeutic agents to stop the continued emergence

of antibiotic resistance. The exact antimicrobial
mechanism of cathelicidin is not clearly compre-
hended. However, it is generally believed that its
physical interactions with the negatively charged
microbial membrane (phospholipids) resulting in
membrane disruption is mainly responsible for its
antimicrobial activity.
Here we report the molecular cloning, identification
and functional analysis of the cathelicidin from donkey
(Equus asinus). Two cathelicidin-encoding cDNAs, one
having a complete coding region (EA-CATH1) and
the other only covering the mature peptide region
(EA-CATH2), were cloned from the constructed lung
cDNA library of donkey. The deduced mature antimi-
crobial peptide EA-CATH1 was synthesized, and an
array of functional activities, including antimicrobial,
haemolytic and erythrocyte haemagglutination, were
examined. Furthermore, the bacterial killing kinetics
and factors related to antimicrobial activity (serum sta-
bility, pH value) were also investigated. To better
understand the mechanism of bactericidal action, the
solution structure of EA-CATH1 was determined using
CD spectroscopy and the effects on bacterial cell
morphology were tested using scanning electron
microscopy (SEM).
Results and Discussion
Identification and characterization of donkey
cathelicidins
We simultaneously constructed cDNA libraries of
jugular lymph, penis, testis, lung, liver, spleen and bone

marrow from donkey. Among them, the lung cDNA
library was of the best quality, from which some posi-
tive clones containing an insert of 555 bp were identi-
fied and isolated. The nucleotide sequence of cDNA
(from the start codon) (GenBank accession FJ803910)
and deduced amino acid sequence of EA-CATH1 pre-
cursor are shown in Fig. 1. Meanwhile, a clone with an
insert of 450 bp was also sequenced, but lacked a signal
peptide and partial cathelin domain. Using BLAST
it was found that the cDNA coding region of
EA-CATH1 displayed maximal 93% identity to the
myeloid cathelicidin 2 (ECATH-2) of Equus caballus
(GenBank accession NM001081869). The EA-CATH1
precursor was composed of 155 amino acid residues,
including a predicted signal peptide, a conserved
cathelin domain and the mature antimicrobial peptide
EA-CATH1 (Fig. 1). Similar to other cathelicidins
identified to date, prepro-EA-CATH1 also contained
four cysteine residues in the conserved region [12]
(Fig. 2).
The processing of cathelicidin to generate mature
antimicrobial peptides has been studied both in vitro
and in vivo. Upon stimulation, the prepropeptide is
processed to release the cathelin domain and the
mature peptide. Elastase is generally considered to be
responsible for such processing in fish, bird and mam-
mals. Valine and alanine represent the most common
elastase-sensitive residues [13]. Here, the valine (130) of
prepro-EA-CATH1 is assumed to be the processing
site by donkey elastase-like protease. Thereby, two

mature peptides were predicted: EA-CATH1 (25 amino
acids), KRRGSVTTRYQFLMIHLLRPKKLFA, and
EA-CATH2 (26 amino acids), KGRGSETTRYQFV-
PVHFFPWNKLSDF. Using BLAST they were found
to be quite divergent from other mammalian cathelici-
dins, even those characterized from horse. Analysis
using the protparam tool ( />protparam.html) showed that the theoretical pI ⁄ Mw
for EA-CATH1 and EA-CATH2 are 12.02 ⁄ 3060.75
and 9.70 ⁄ 3144.54, respectively. EA-CATH1 is a basic
peptide smaller than most of the other cathelicidins
identified to date. It comprises seven basic residues
(four arginine and three lysine) with a net charge of 7.
Thus, EA-CATH1 would be readily attracted by and
adhere to the negatively charged bacterial surface to
exert its potent antimicrobial activity.
Characterization of cathelicidin from Equus asinus Z. Lu et al.
2330 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS
Phylogenetic relationship between EA-CATH1
and other cathelicidins
Multisequence alignment was performed on the basis
of the full sequence of all cathelicidins. A condensed
multifurcating tree was constructed emphasizing the
reliable portion of pattern branches without consider-
ing the exact distance between each peptide. Thus,
the branch lengths of the condensed tree are not
proportional to the number of amino acid mutations.
The built phylogenetic tree revealed that vertebrate
cathelicidins are split into two major clusters, and
the sister group is represented by CATH37 from
hagfish in a separated clade, which was potentially

considered as an ancient member in the cathelicidin
evolution. The second cluster is divided into two
major groups: one represented by Atlantic cod, rain-
bow trout; the other represented by snake cathelici-
dins, avian fowlicidins and the most divergent
mammalian cathelicidin families. Supported by a
bootstrap value of 79%, EA-CATH1 was clustered
with horse eCATH-1 and -3 (Fig. 3).
Antimicrobial activity and bacteria killing kinetics
Putatively mature EA-CATH1 was commercially syn-
thesized and purified to > 95% purity. As listed in
Table 1, EA-CATH1 showed broad-spectrum antimi-
crobial activities against the tested micro-organisms,
especially clinically isolated drug-resistant strains. In
all antimicrobial assays, LL-37 characterized from
human was used as the positive control. It is one of
the most extensively studied cathelicidins so far. Com-
pared with minimal inhibitory concentrations (MICs)
of LL-37, EA-CATH1 showed much stronger antibac-
terial potency. Among all 32 strains, Gram-positive
bacterial strains were much more sensitive to
EA-CATH1 than Gram-negative strains and fungus,
with most MIC values in the range of 0.6–4.7 lgÆmL
)1
(Table 1). EA-CATH1 even had a potent killing effect
on the strains that were totally resistant to the conven-
tional antibiotic drugs, e.g. Enterococcus faecium
(IS091299) (MIC 9.4 lgÆmL
)1
). EA-CATH1 showed

the strongest antimicrobial activity against Staphylo-
coccus aureus ATCC2592 and S. haemolyticus 092401
with MICs as low as 0.6 lgÆmL
)1
. For clinically iso-
lated S. aureus and Nocardia asteroids, the MICs were
both determined to be only 1.2 lgÆmL
)1
. Interestingly,
we also tested the antimicrobial activity of EA-CATH1
against Propionibacterium acnes, one kind of bacteria
bothering a large population all over the world.
EA-CATH1 also had a fairly small MIC of
4.7 lgÆmL
)1
. However, half of the Gram-negative bac-
teria tested seemed not to be very sensitive to
EA-CATH1 and LL-37 performed even worse.
The killing kinetics of EA-CATH1 were examined
using a colony counting assay, with ampicillin as the
positive control. As listed in Table 2, EA-CATH1
exerted antibacterial activity in a faster kinetics than
ampicillin. It could rapidly kill S. aureus (ATCC2592),
with the maximum killing occurring at less than 0.5 h
(versus 2 h for ampicillin) at 10· MIC; 1 h (versus 3 h
Fig. 1. The cDNA sequence encoding
EA-CATH1 and the predicted prepropeptide
sequence. The signal peptide predicted by
SIGNALP 3.0 is shaded in grey. The putative
mature peptide of EA-CATH1 is boxed. The

stop codon is indicated by an asterisk. The
3¢- UTR is in lowercase letters. The potential
polyadenylation signal (aaaaataaa) is
underlined.
Z. Lu et al. Characterization of cathelicidin from Equus asinus
FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2331
for ampicillin) at 5· MIC and 2 h (versus 6 h for
ampicillin) at 1· MIC. The antibacterial activity
proved to be lethal for S. aureus ATCC2592. Staphylo-
coccus aureus was not capable of resuming growth on
agar plates after a 2 h treatment with concentrations
above the corresponding MICs. In contrast, ampicillin
could not clean the bacteria within 2 h. EA-CATH1 of
5· MIC killed micro-organisms almost five times faster
than 1· MIC (Table 2).
Secondary structures of EA-CATH1 and the
effects on bacterial cell morphology
The CD spectrum of EA-CATH1 in water showed a
negative band at 200 nm, indicating a random coil
conformation. In a membrane-mimetic solvent such as
50% trifluoroethanol ⁄ water, the presence of one posi-
tive band (190 nm) and two negative dichroic bands at
208 and 222 nm are consistent with the a-helical con-
formation (Fig. 4). The current result is in good agree-
ment with the online prediction by GOR IV (http://
npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_
gor4.html), which showed a 36% a-helical peptide
(Y9-L17) in the middle, a 56% random coil (K1-R8,
R18-K22, A25) on both sides of the a-helix and two
amino acid extended stands (L23, F24) close to the

C-terminus. The a-helical structure of most active
cathelicidin peptides is thought to be responsible for
the formation of pores in the membranes of target
organisms, thus disrupting metabolic activity [14]. This
is also approved by LL-37 [15]. Its helical, oligo-
meric conformation is required for potent antibacterial
Fig. 2. Multiple sequence alignment of EA-CATH1 with representative cathelicidins; conserved residues are shaded. The four conserved
cysteine residues in cathelin domain are framed. Each mature cathelicidin is underlined. Ea, Equus asinus (donkey); Ec, Equus caballus
(horse) [12]; Clf, Canis lupus familiars (dog) [27]; Bt, Bos taurus (cattle) [28]; Oa, Ovis aries (sheep) [29]; Ch, Capra hircus (goat) [30]; Ss,
Sus scrofa (pig) [31]; Hs, Homo sapiens (human) [7]; Oc, Oryctolagus cuniculus (rabbit) [32]; Mm, Mus musculus (mouse) [33]; Cp, Cavia
porcellus (guinea pig) [34]; Gg, Gallus gallus (chicken) [8]; Me, Macropus eugenii (tammar wallaby) [35]; Bf, Bungarus fasciatus (snake) [22].
Characterization of cathelicidin from Equus asinus Z. Lu et al.
2332 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS
98
99
64
97
98
92
100
100
100
95
73
70
94
81
78
92
83

56
98
56
90
66
100
70
83
79
98
69
63
100
61
78
98
100
100
100
98
Fig. 3. Phylogenetic analysis of representa-
tive vertebrate cathelicidins. The phyloge-
netic dendrogram was constructed using
the neighbour-joining method based on the
proportion difference of aligned amino acid
sites of the full sequence of prepropeptide.
Only bootstrap values > 50% (expressed as
a percentage of 1000 bootstrap samples
supporting the branch) are shown at
branching points. The bar indicates the

branch length.
Z. Lu et al. Characterization of cathelicidin from Equus asinus
FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2333
activity. The CD result supports the conception that
EA-CATH1 probably kills bacteria through membrane
disruption.
A generally acknowledged antimicrobial mechanism
of cathelicidin is its physical interactions with the neg-
atively charged microbial membrane, followed by
membrane lysis [16]. Such an interaction is often
directly correlated with the extent of antibacterial
activity and makes it hard to develop resistance [17].
In the present study, the effects of EA-CATH1 on the
cellular morphology of S. aureus were observed by
SEM. Control cells with no peptide treatment exhib-
ited a normal shape and smooth surfaces (Fig. 5A). In
contrast, treatment with EA-CATH1 for 30 min
severely disrupted the cell wall and cell membrane of
S. aureus (Fig. 5B–D). During treatment, the bacterial
cells appeared to have a rough surface, with crimpled
and bent morphologies (Fig. 5B–D), and were then
finally lysed.
Haemolysis, serum stability and the effect of pH
on antimicrobial activity
A big problem commonly associated with clinical
applications of cathelicidins is their haemolysation of
mammalian cells. However, the good thing is that the
dose of cathelicidin resulting in haemolysis is often
much higher than the MIC. The haemolytic capability
Table 1. Antimicrobial activity of EA-CATH1. These concentrations represent the mean values of three independent experiments performed

in duplicate. ND, no detectable activity in the inhibition zone assay at a dose of 2 mgÆmL
)1
; > 100, detectable antimicrobial activity in the
inhibition zone assay, but did not totally inhibit cell growth in liquid medium at a dose up to 100 lgÆmL
)1
; IS, clinically isolated strain; DRa,
drug resistance for ceftazidime, cefoperazone and aztreonam; DRb, drug resistance for compound sulfamethoxazole, erythromycin, ciproflox-
acin and penicillin.
Micro-organism
MIC (lgÆmL
)1
)
EA-CATH1 LL-37 Ampicillin Kanamycin
Gram positive
Staphylococcus aureus (IS) 1.2 18.8 4.7 75
Staphylococcus aureus ATCC2592 0.6 4.7 2.4 4.7
Staphylococcus haemolyticus (092401) (IS, DRa) 0.6 ND 0.3 ND
Nocardia asteroids (IS) 1.2 18.8 1.2 75
Enterococcus faecium (IS091299) 9.4 18.8 ND ND
Propionibacterium acnes ATCC11827 4.7 > 100 1.2 2.4
Gram negative
Klebsiella oxytoca (IS) > 100 ND ND ND
Aeromonas sobria (IS) 9.4 18.8 ND ND
Acinetobacter baumannii 092178 (IS, DRb) 4.7 ND 37.5 2.4
Acinetobacter baumannii 092373 (IS) 9.4 ND ND ND
Stenotrophomonas maltophilia (IS) 9.4 18.8 ND ND
Pseudomonas aeruginosa ATCC27853 > 100 ND ND ND
Pseudomonas aeruginosa 091411(IS) 18.8 > 100 ND ND
Pseudomonas aeruginosa 091412 (IS) > 100 ND ND ND
Pseudomonas aeruginosa 091413 (IS) > 100 ND > 100 75

Escherichia coli 091335 (IS) 75 ND ND ND
Escherichia coli ATCC25922 ND ND 9.4 9.4
Escherichia coli 090223 (IS) ND ND 75 2.4
Serratia marcescens 091379 (IS) ND ND ND ND
Klebsiella pneumoniae 091372 (IS) > 100 ND ND ND
Klebsiella pneumoniae 091373 (IS) > 100 ND ND 2.4
Klebsiella pneumoniae 091400 (IS) ND ND ND ND
Proteus vulgaris (IS) > 100 > 100 1.2 4.7
Proteus mirabilis (IS) > 100 ND 2.4 4.7
Salmonella typhi 091408 (IS) ND ND ND 18.8
Fungi
Candida albicans 092251 (IS) 9.4 > 100 4.7 18.8
Candida albicans ATCC2002 9.4 9.4 0.6 1.2
Slime mould 4.7 18.8 2.4 75
Candida glabrata 090902 (IS) > 100 ND ND ND
Candida tropicalis 092422 (IS) > 100 ND ND ND
Cryptococcus neoformans (IS) ND ND ND ND
Characterization of cathelicidin from Equus asinus Z. Lu et al.
2334 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS
of EA-CATH1 was tested using freshly prepared
human erythrocytes. The result indicated that
EA-CATH1 (20 lgÆmL
)1
) had almost no haemolytic
activity (1.8%) on human red blood cells in a dose
much higher than the MIC. Thus, EA-CATH1 showed
considerable selectivity for micro-organisms over mam-
malian cells in vitro.
The serum stability of EA-CATH1 was also exam-
ined; the results are listed in Table 3. To our surprise,

after incubating with 90% fresh human serum for up
to 72 h, EA-CATH1 still retained strong antimicrobial
activity against S. aureus, much longer than other
Table 2. Bacterial killing kinetics of EA-CATH1.
Time
cfu (Staphylococcus aureus ATCC2592)
0 min 10 min 30 min 1 h 1.5 h 2 h 3 h 6 h
EA-CATH1(·1 MIC
a
)63 44 21 9 1 0 0 0
EA-CATH1(·5 MIC) 57 22 3 0 0 0 0 0
EA-CATH1(·10 MIC) 54 5 0 0 0 0 0 0
Ampicillin (·1 MIC
b
)67 62 42 19 9 4 1 0
Ampicillin (·5 MIC) 61 46 23 6 3 1 0 0
Ampicillin (·10 MIC) 60 37 13 4 1 0 0 0
Water 68 87 113 177 264 432 1280 17 175
cfu (Acinetobacter baumannii 092178 IS)
EA-CATH1(·1 MIC
c
)61 55 49 38 28 12 1 0
EA-CATH1(·5 MIC) 57 46 39 22 5 0 0 0
EA-CATH1(·10 MIC) 55 41 33 15 0 0 0 0
Water 62 81 148 234 355 591 1706 22 724
a
EA-CATH1 MIC to S. aureus ATCC2592 0.6 lgÆmL
)1
;
b

ampicillin MIC to S. aureus ATCC2592 2.4 lgÆmL
)1
;
c
EA-CATH1 MIC to A. baumannii
(092178 IS) 4.7 lgÆmL
)1
.
100
EA-CATH1
40
60
80
Water
TFE 50%
–20
0
20
CD [mdeg]
–80
–60
–40
190 200 210 220 230 240 250
Wavelen
g
th [nm]
Fig. 4. CD analysis of EA-CATH1 in trifluoroethanol ⁄ water (50% v ⁄ v).
9051910 1.0 kV 7.4 mm x20.0k SE(M) 5/20/2009 09:40
905197 1.0 kV 7.6 mm x20.0k SE(M) 5/20/2009 10:34
905198 1.0 kV 7.6 mm x20.0k SE(M) 5/20/2009 10:13

905197 1.0 kV 7.6 mm x20.0k SE(M) 5/20/2009 10:31
1xMIC
10xMIC
10xMIC
2.00 µm
2.00 µm
2.00 µm
2.00 µm
AB
CD
Fig. 5. SEM of Staphylococcus aureus trea-
ted with EA-CATH1. (A) Control S. aureus;
(B) S. aureus treated with EA-CATH1 at 1·
MIC; (C, D) S. aureus treated with
EA-CATH1 at 10· MIC.
Z. Lu et al. Characterization of cathelicidin from Equus asinus
FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2335
cathelicidins [18]. Such extraordinary stability in serum
implies the potential of EA-CATH1 for systemic thera-
peutic applications. More interestingly, during the first
3 h after adding EA-CATH1 to serum, the MIC
(0.6 lgÆmL
)1
) against S. aureus was lower by half than
in water. This might be due to the antibacterial activity
of serum proteins, which lately has been given a lot of
attention. Or, possibly, our peptide is highly serum
protein bound, which could lead to the conformational
change (a more helical structure) and would explain
the lower MICs in the presence of the serum. The

MICs of EA-CATH1 incubating with human serum
for 3–12, 24–48 and 60–72 h were 1.2, 4.7 and
9.4 lgÆmL
)1
, respectively.
The effects of pH on the antimicrobial activity of
EA-CATH1 were tested (Table 4). Clearly, in the pH
range of 5.0–9.0, the acidic pH (5.0–7.0) benefited the
antimicrobial effect against S. aureus (Gram positive),
whereas Acinetobacter baumannii (Gram negative) and
Candida albicans (fungus) were more sensitive to
EA-CATH1 at the basic pH (7.0–9.0). Among the
three strains, the MIC for C. albicans was influenced
most, varying from 2.4 to 18.8 lgÆmL
)1
under a corre-
sponding pH from 9.0 to 5.0. At optimal pH values
around 7.0, EA-CATH1 showed the strongest antimi-
crobial activities with the lowest MICs. The explana-
tion for such pH-dependent activity is the pH-induced
structural changes in peptide conformation. The a-heli-
cal structure is thought to be important for the antimi-
crobial activity of cathelicidins [14], and its content is
usually unchanged over the neutral pH range, but is
drastically reduced at higher or lower pH values.
Thereby, the pH-induced peptide unfolding may con-
tribute to the reduced activity of EA-CATH1 at acidic
or basic pH values. The assay against C. albicans
might have involved certain inevitable error resulting
in a slightly higher optimal pH (8.0). The other expla-

nation is that EA-CATH1 might exert its antifungal
activity through the formation of reactive oxygen spe-
cies [19]. This process is irrelevant to peptide solution
structure, thus in turn irrelevant to pH.
Erythrocytes haemagglutination activity
EA-CATH1 had no detected haemagglutination activ-
ity on fresh rabbit erythrocytes in the assay. However,
in the presence of CaCl
2
, it could exert an agglutina-
tion activity with the minimum concentration of
50 lgÆmL
)1
(16.3 lm). So far, significant peptide-
induced haemagglutination has been observed for
certain cathelicidins, such as LL-37 (‡ 25 lm) and
indolicidin (‡ 100 lm) [14]. It has been proposed that
the bacteria secreted or membrane-bound polyproteins
can bind to, agglutinate and lyse local host erythro-
cytes [20]. Thus, the cationic cathelicidins might poten-
tiate an inhibition against the electrostatic interaction
between the bacterial polyproteins and the haemag-
glutinin binding domains on the erythrocyte surface
[21]. It has been reported that antimicrobial
peptides, including cathelicidin LL-37, were effective in
disrupting Porphyromonas gingivalis-induced haema-
gglutination among erythrocytes [22]. Therefore, the
haemagglutination ability of EA-CATH1 in the pres-
ence of CaCl
2

makes it a good drug candidate to
potentially restrict bacterial colonization and spread by
the perturbation of bacterial polyproteins.
In summary, in the present work, EA-CATH1
was identified by molecular cloning as a member of
Table 3. Stability of EA-CATH1 in human serum.
MIC (lgÆmL
)1
)
Time (h) 036122436486072
Clinically isolated Staphylococcus aureus 0.6 1.2 1.2 1.2 4.7 4.7 4.7 9.4 9.4
Table 4. Antimicrobial activity of EA-CATH1 in 150 mM NaCl ⁄ P
i
at different pH values (mean values of three independent experiments
performed in duplicate). IS, clinically isolated strain; DRb, drug resistance for compound sulfamethoxazole, erythromycin, ciprofloxacin and
penicillin. –, S. aureus (IS) did not grow.
Micro-organism
MIC (lgÆmL
)1
)
Water pH 5 pH 6 pH 7 pH 8 pH 9
Staphylococcus aureus (IS) 1.2 – 0.6 0.6 1.2 1.2
Acinetobacter baumannii 092178(IS, DRb) 4.7 9.4 2.4 4.7 4.7 4.7
Candida albicans ATCC2002 9.4 18.8 9.4 4.7 2.4 2.4
Characterization of cathelicidin from Equus asinus Z. Lu et al.
2336 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS
cathelicidin-derived antimicrobial peptides from don-
key (E. asinus). The nucleotide and deduced amino
acid sequences of prepro-EA-CATH1 were compar-
atively conserved among mammalian cathelicidin

families. The chemically synthesized EA-CATH1 has
broad-spectrum potent antibacterial activity, but no
haemolytic activity in high doses, implying a promis-
ing therapeutic potential. In addition, the human
serum stability and haemagglutination capacity of
EA-CATH1 makes it an excellent candidate for the
development of novel antimicrobial and antisepsis
agents. The results of a pH-dependency assay coupled
with killing kinetics may offer important data for
clinical studies.
Materials and methods
Collection of tissues
Tissue samples of an adult male donkey were collected
from Beijing Hongfa Donkey Meat Processing Plant
(Beijing, China), including lung, spleen, liver, jugular
lymph, testis, penis and bone marrow. The collection proce-
dure was according to either routine management of the
farm animals or surplus from other approved research pro-
tocols. Tissues were dissected and frozen immediately in
liquid nitrogen until used.
Molecular cloning of cathelicidin and
phylogenetic tree construction
Total RNA was extracted from each tissue collected using
the RNeasy Mini Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s instructions. PCR-based cDNA was
synthesized using the CreatorÔ SMARTÔ cDNA library
construction kit (Clontech, Palo Alto, CA, USA) as
described by the manufacturer. The first-strand cDNA was
synthesized using PowerScript reverse transcriptase with the
SMART

TM
IV oligonucleotide primer 5¢-AAGCAGTGGT
ATCAACGCAGAGTGGCCATTACGGCCGGG-3¢ and
the CDS III ⁄ 3¢ PCR primer 5¢-ATTCTAGAGGCCGA
GGCGGCCGACA TGT(30)N
-1
N-3¢ (N = A, G, C or T;
N
-1
= A, G or C). The second strand was amplified
using Advantage DNA polymerase from Clontech with the
5¢ PCR primer 5¢-AAGCAGTGGTATCAACGCAGAGT-3¢
and the CDS III ⁄ 3¢ PCR primer.
According to the conserved signal peptide domain of pre-
viously characterized horse cathelicidin cDNA [23], two
sense primers P1 (5¢-GGACCATGGAGACCCAGAGG-3¢)
and P2 (5¢-ATGGAGACCCAGAGGGACAGTT-3¢) were
designed from 5¢-UTR and a highly conserved domain-
encoding part of the signal peptide of horse cathelicidin
cDNAs and coupled with CDS III ⁄ 3¢ PCR primer. The half
nested PCR conditions involved two sections. First section:
94 °C for 1 min; 25 cycles of 94 °C for 30 s, 60 °C for 30 s,
72 °C for 60 s; followed by a final extension at 72 °C for
10 min. Second section: 94 °C for 5 min; 30 cycles of 94 °C
for 20 s, 58 °C for 20 s, 72 °C for 45 s; followed by a final
extension at 72 °C for 10 min. The PCR product was purified
by gel electrophoresis, cloned into pGEM-T vector (Pro-
mega, Madison, WI, USA). DNA sequencing was performed
on an Applied Biosystems DNA sequencer, model ABI
PRISM 377 (Perkin Elmer Corp., Norwalk, CT, USA).

The phylogenetic tree was constructed with the neigh-
bour-joining method using clustalw (version 1.8). Multi-
cathelicidin sequences aligned were obtained from the
protein database at the National Center for Biotechnology
Information.
CD spectroscopy
The peptide used for the bioactivity test and CD spectros-
copy was synthesized by the peptide synthesizer GL
Biochem (Shanghai, China), and purified to > 95% purity.
To investigate the secondary structure of EA-CATH1, CD
spectroscopy was performed using a Jasco J-715 spectro-
photometer. Samples with a constant peptide concentration
of 0.5 mgÆmL
)1
were prepared in two different solvents,
water and 50% (v ⁄ v) trifluoroethanol ⁄ water, and added
in a quartz optical cell with a path length of 0.5 mm at
25 °C. The spectra were averaged over three consecutive
scans, followed by subtraction of the CD signal of the
solvent.
Antimicrobial assay and bacteria killing kinetics
In total, 31 standard (purchased commercially) and clini-
cally isolated bacterial and fungal strains (obtained from a
local hospital) were used for the antimicrobial assays
(Table 1). The assay was conducted as described previously
[24]. The MIC was measured using the standard micro-
dilution broth method in a 96-well microtitre plate. Serial
dilutions (50 lL) of the peptides in Mueller–Hinton broth
(MH) were prepared in 96-well microtitre plates and mixed
with 50 lL bacteria inoculums in MH [1 · 10

6
colony-
forming units (cfu)ÆmL
)1
]. The human cathelicidin LL-37
and the antibiotics ampicillin and kanamycin were used as
positive controls. The microtitre plate was incubated at
37 °C for 18 h for bacteria and 48 h for fungal strains and
absorbance was measured at 595 nm using a microtitre
plate spectrophotometer. MIC was defined as the lowest
concentration of peptide that completely inhibits growth of
the microbe determined by visual inspection or spectro-
photometrically the growth percentage was less than 5%
compared with that of the negative control.
The bactericidal effects of EA-CATH1 against S. aureus
ATCC2592 (1 · 10
6
cfuÆmL
)1
) and A. baumannii (1 ·
10
6
cfuÆmL
)1
) were tested at 1, 5 and 10· corresponding
Z. Lu et al. Characterization of cathelicidin from Equus asinus
FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2337
MICs, with ampicillin as the positive control. Fresh colo-
nies of the bacteria were cultured overnight to log phase,
measured absorbance at 600 nm (A

600
)is 8 · 10
8
cfuÆmL
)1
and then diluted with fresh MH to 1 · 10
6
cfuÆmL
)1
. EA-CATH1 was added to the bacterial suspen-
sion, achieving the final sample concentration to 1, 5 and
10 · corresponding MICs. The mixture was incubated at
37 °C. Colony counting was performed at 0 min, 10 min,
30 min, 1 h, 1.5 h, 2 h, 3 h and 6 h [24]. At each time
point, 1 lL mixture was diluted with MH to 1 mL, then
50 lL diluted bacterial suspension was plated out at 37 ° C
for 12 h before colony counting.
SEM
A log phase culture (1 · 10
6
cfuÆmL
)1
)ofS. aureus
(ATCC2592) was incubated with EA-CATH1 (1 · ,
10 · MIC) at 37 °C for 30 min. Aliquots of the cultures
were fixed with 6% glutaraldehyde solution for 4 h. The
bacteria were then centrifuged (300 g for 10 min) and
washed with 0.1 m phosphate-buffered saline (NaCl ⁄ P
i
), pH

7.2. The pellets were then fixed in 1% osmium tetroxide in
0.1 m NaCl ⁄ P
i
, pH 7.2 for 1 h. The cells were rinsed with
the same buffer and dehydrated in a graded series of etha-
nol and then frozen in liquid nitrogen-cooled tert-butyl
alcohol and vacuum dried overnight. The samples were
mounted on to aluminium stubs. After sputter coating with
gold, they were analysed using a Hitachi S-4800 SEM.
Haemolysis, serum stability and the effect of pH
on antimicrobial activity
Haemolysis assays were conducted as previously described
[25]. The EA-CATH1 of 20 lgÆmL
)1
was incubated with
washed human erythrocytes at 37 °C for 30 min and centri-
fuged at 1000 g for 5 min. Absorbance of the supernatant
was measured at 540 nm. Triton X-100 (1% v ⁄ v) was used
to determine the maximal haemolysis. The experiment was
repeated three times. The serum stability of EA-CATH1
(2 mgÆmL
)1
) was examined by incubating with 90% freshly
prepared human serum at 37 °C for 0, 3, 6, 12, 24, 36, 48,
60 and 72 h. The MIC was then recorded at each time
interval. EA-CATH1 was dissolved in 150 mm NaCl ⁄ P
i
(sterilized by filter) at pH 4, 5, 6, 7, 8 and 9. The MICs of
EA-CATH1 on Gram-positive bacterium S. aureus, Gram-
negative bacterium A. baumannii and fungus C. albicans

(ATCC2002) cultured in MH were then tested.
Erythrocyte haemagglutination assay
Fresh intact rabbit erythrocytes were prepared as previously
described [26]. Assays were performed in 96 U-well micro-
titre plates. The haemagglutinating activity of EA-CATH1
was determined by a two-fold serial dilution procedure
using rabbit erythrocytes. The haemagglutination titre was
defined as the reciprocal of the highest dilution exhibiting
haemagglutination. To examine the divalent cation effect,
20 mm Tris ⁄ HCl (pH 7.5) with or without 50 mm CaCl
2
was used as the assay buffer.
Acknowledgement
We thank the editor and four anonymous reviewers
for their helpful comments on the manuscript. This
work was supported by grants from the Chinese
National Natural Science Foundation (30900240).
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