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

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 strai

Equus asinus

Zekuan Lu1*, Yipeng Wang2,3*, Lei Zhai1, Qiaolin Che3, Hui Wang1, Shuyuan Du1, Duo Wang1, Feifei Feng1,2, Jingze Liu1, Ren Lai3and Haining Yu1,2

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: rlai@mail.kiz.ac.cn

H Yu, College of Life Sciences, Hebei

Normal University, Shijiazhuang, Hebei

050016, China

Fax ⁄ Tel: +86 311 86268842

E-mail: yuhaining@dlut.edu.cn

*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 CaCl2, 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.

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 (http://au.expasy.org/tools/ 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

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.

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 negaposi-tive 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].

98 99

98 92

100

100 100 95

73

70

94

81 78 92 83 56

98 56

90 66

100 70

83 79 98 69

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.

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)

Gram positive

Gram negative

Fungi

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)

EA-CATH1(·1 MIC a

cfu (Acinetobacter baumannii 092178 IS)

EA-CATH1(·1 MIC c

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

–80

–60

–40

Wavelength [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

2.00 µm

2.00 µm

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.

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)1under 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 CaCl2, it could exert an agglutina-tion activity with the minimum concentraagglutina-tion 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 CaCl2 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)

Table 4 Antimicrobial activity of EA-CATH1 in 150 m M 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)

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 Creator SMART 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

SMARTTM IV oligonucleotide primer 5¢-AAGCAGTGGT

ATCAACGCAGAGTGGCCATTACGGCCGGG-3¢ and

the CDS III⁄ 3¢ PCR primer 5¢-ATTCTAGAGGCCGA

GGCGGCCGACA TGT(30)N-1N-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:

94C for 1 min; 25 cycles of 94 C for 30 s, 60 C for 30 s,

72C for 60 s; followed by a final extension at 72 C for

10 min Second section: 94C for 5 min; 30 cycles of 94 C for 20 s, 58C for 20 s, 72 C for 45 s; followed by a final extension at 72C 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

25C 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· 106

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

37C 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· 106

cfuÆmL)1) and A baumannii (1·

106cfuÆmL)1) were tested at 1, 5 and 10· corresponding

MICs, with ampicillin as the positive control Fresh

colo-nies of the bacteria were cultured overnight to log phase,

measured absorbance at 600 nm (A600) is  8 · 108

cfuÆmL)1 and then diluted with fresh MH to 1· 106

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

37C 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 37C

for 12 h before colony counting

SEM

A log phase culture (1· 106

cfuÆmL)1) of S 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⁄ Pi), pH

7.2 The pellets were then fixed in 1% osmium tetroxide in

0.1 m NaCl⁄ Pi, 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 37C 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 37C 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⁄ Pi

(sterilized by filter) at pH 4, 5, 6, 7, 8 and 9 The MICs of

EA-CATH1 on 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 CaCl2

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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