Gene cloning, expression and characterization of avian ...ir.yic.ac.cn/bitstream/133337/4869/1/Gene cloning... · Gene cloning, expression and characterization of avian cathelicidin

Gene cloning, expression and characterization of aviancathelicidin orthologs, Cc-CATHs, from Coturnix coturnixFeifei Feng1,2,*, Chen Chen3,*, Wenjuan Zhu2, Weiyu He1, Huijuan Guang2, Zheng Li2, Duo Wang1,Jingze Liu1, Ming Chen5, Yipeng Wang4 and Haining Yu1,2

1 College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei, China

2 School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning, China

3 College of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong, Shaanxi, China

4 Biological Resources Laboratory, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shandong, China

5 Department of Nephrology, Teaching Hospital of Chengdu University of Traditional Chinese Medical, Chengdu, China

Introduction

A large group of gene-encoded antimicrobial peptides

has been discovered in almost all species of organism,

forming a first line of host defense against environmen-

tal microorganisms [1–3]. This group is classified into

several families, including cathelicidin, liver-expressed

antimicrobial peptide or hepcidin, histatin and defensin

[4–8]. At the chemical level, the defensins and hepci-

dins comprise small peptides that are usually rich in

cysteine [5–7], whereas histatins and cathelicidin-

derived antimicrobial peptides are mostly linear mole-

cules without disulfide bridges [8].

Cathelicidins represent a relatively young family of

endogenous antibiotics first discovered in bovine neu-

trophils [9]. Subsequently, numerous cathelicidins have

Keywords

cathelicidin; Coturnix coturnix; expression;

molecular cloning; structure and function

Correspondence

H. Yu or Y. Wang, College of Life Sciences,

Hebei Normal University, Shijiazhuang,

Hebei 050016, China; Biological Resources

Laboratory, Yantai Institute of Coastal Zone

Research, Chinese Academy of Sciences,

Yantai, Shandong 264003, China

Fax: +86 311 86268842

Tel: +86 311 86268842

E-mail: [email protected]; [email protected]

*These authors contributed equally to this

work

(Received 7 November 2010, revised 20

February 2011, accepted 23 February 2011)

doi:10.1111/j.1742-4658.2011.08080.x

Cathelicidins comprise a family of antimicrobial peptides sharing a highly

conserved cathelin domain, which play a central role in the early innate

host defense against infection. In the present study, we report three novel

avian cathelicidin orthologs cloned from a constructed spleen cDNA

library of Coturnix coturnix, using a nested-PCR-based cloning strategy.

Three coding sequences containing ORFs of 447, 465 and 456 bp encode

three mature antimicrobial peptides (named Cc-CATH1, 2 and 3) of 26,

32 and 29 amino acid residues, respectively. Phylogenetic analysis indi-

cated that precursors of Cc-CATHs are significantly conserved with

known avian cathelicidins. Synthetic Cc-CATH2 and 3 displayed broad

and potent antimicrobial activity against most of the 41 strains of bacte-

ria and fungi tested, especially the clinically isolated drug-resistant strains,

with minimum inhibitory concentration values in the range 0.3–2.5 lM for

most strains with or without the presence of 100 mM NaCl. Cc-CATH2

and 3 showed considerable reduction of cytotoxic activity compared to

other avian cathelicidins, with average IC50 values of 20.18 and 17.16 lM,

respectively. They also exerted a negligible hemolytic activity against

human erythrocytes, lysing only 3.6% of erythrocytes at a dose up to

100 lgÆmL)1. As expected, the recombinant Cc-CATH2 (rCc-CATH2)

also showed potent bactericidal activity. All these features of Cc-CATHs

encourage further studies aiming to estimate their therapeutic potential as

drug leads, as well as coping with current widespread antibiotic resis-

tance, especially the new prevalent and dangerous ‘superbug’ that is resis-

tant to almost all antibiotics.

Abbreviations

IPTG, isopropyl thio-b-D-galactoside; MH, Mueller–Hinton; MIC, minimum inhibitory concentration; rCc-CATH2, recombinant Cc-CATH2.

FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 1573

been identified from mammals, including humans,

monkey, mouse, rat, rabbit, guinea pig, pig, cattle,

sheep, goat and horse [9–14]. Cathelicidins have also

been reported in bird and fish species, such as fowlici-

din-1, -2, -3, B1 and myeloid antimicrobial peptide 27

from chicken [15,16], as well as Atlantic hagfish (Myx-

ine glutinosa), rainbow trout (Oncorhynchus mykiss)

and Atlantic salmon (Salmo salar). Hagfish cathelici-

dins were considered as ancient members of the cath-

elicidin family [17–19]. Recently, cathelicidin sequences

from reptile species such as Naja atra, Bungarus fascia-

tus and Ophiophagus hannah were also obtained

[20,21]. Generally, cathelicidins are characterized by a

highly conserved N-terminal signal peptide (approxi-

mately 30 residues) and cathelin domain (99–114

residues long), followed by a highly heterogeneous

C-terminal mature peptide (12–100 residues) [4,22,23].

In addition to their primary antimicrobial activities,

cathelicidins are also found to be actively involved in

various phases of host defense, such as the induction

of angiogenesis, the promotion of wound healing, and

chemotaxis for neutrophils, monocytes, mast cells and

T cells, as well as the inhibition of apoptosis [1,24,25].

Consistent with their critical role in the host innate

immune system, the aberrant expression of cathelici-

dins is often associated with various disease processes

[26,27].

In the present study, the gene cloning and character-

ization of three avian cathelicidin orthologs, namely

Cc-CATH precursors from Coturnix coturnix, is

reported, and the relationship between quail cathelici-

dins and other known vertebrate cathelicidins is ana-

lyzed. Two of the three cathelicidin-derived

antimicrobial peptides, Cc-CATH2 and 3, were chemi-

cally synthesized and their antimicrobial activities were

examined. They were found to kill Gram-positive and

-negative bacteria, as well as fungi, in a salt-indepen-

dent manner, with almost no hemolytic activity and

cytotoxicity. Moreover, recombinant Cc-CATH2

(rCc-CATH2) was produced in Escherichia coli. The

purified rCc-CATH2 maintained its broad and potent

bactericidal activity. The present study may represent

the probation experiment for future industrial, large-

scale production.

Results

Identification and characterization of quail

cathelicidins

Total RNA was extracted from the quail spleen. On

the basis of the end of the 5¢-UTR and the first 20 bp

of the fowlicidin signal peptide cDNA sequence, a set

of primers was designed. Several positive clones con-

taining inserts of 545, 530 and 555 bp were identified

and isolated. The complete nucleotide and translated

amino acid sequences of the three quail cathelicidins

(GenBank accession numbers: GU232858, GU171373

and GU171374 for Cc-CATH1, 2 and 3, respectively)

are shown in Figs 1 and 2. Alignment of three Cc-

CATHs revealed that they share high sequence similar-

ity with each other (Fig. 1) and that Cc-CATH1 and 3

are more closely related, with 93% identity throughout

the entire sequence. Using a blast search, and

unlike the highly divergent mammal cathelicidins even

within the same genus, Cc-CATHs (C. coturnix) were

found to share a high degree of similarity with previ-

ously characterized Pc-CATHs from pheasant [28] and

fowlicidins from chicken (Gallus gallus) [16], particu-

larly in the prosequence region (Figs 1 and 2). The

avian cathelicidins all include a predicted signal

peptide, a conserved cathelin domain and a cationic

C-terminal mature antimicrobial peptide (Fig. 2).

Computational predication with signalp 3.0 software

(http: ⁄ ⁄www.cbs.dtu.dk ⁄ services ⁄SignalP ⁄ ) indicates a

17 amino acid signal peptide located at the N-termi-

nus. Noticeably, four cysteines that are conserved in

the cathelin domain of all cathelicidins identified to

date are also invariantly spaced in Cc-CATHs precur-

sor [11] (Fig. 3).

The processing of cathelicidin to generate mature

antimicrobial peptides has been studied both in vitro

and in vivo [29–31]. The valine of the three prepropep-

tides is assumed to comprise the processing site for

elastase-like protease to generate Cc-CATH1, 2 and 3.

Further assisted by alignment with chicken fowlicidins

and pheasant Pc-CATHs, three mature antimicrobial

peptides were predicted (Fig. 2): Cc-CATH1 (26 amino

acids), RVKRVLPLVIRTVIAGYNLYRAIKRK; Cc-

CATH2 (32 amino acids), LVQRGRFGRFLKKVRR

FIPKVIIAAQIGSRFG; and Cc-CATH3 (29 amino

acids), RVRRFWPLVPVAINTVAAGINLYKAIR

RK. Analysis using the protparam tool (http://au.exp-

asy.org/tools/protparam.html) showed a theoretical

pI ⁄Mw for Cc-CATH1, 2 and 3 of 11.85 ⁄ 3096.85,12.70 ⁄3715.54 and 12.18 ⁄ 3379.11, respectively. Similar

to classic cathelicidins, Cc-CATHs are highly basic at

the C-terminus as a result of the presence of cationic

residues (Arg and Lys), which implies that they would

be readily attracted by and adhere to the negative-

charged bacterial surface, thus explaining its high anti-

microbial potency.

The avian multisequence alignments were performed

on basis of the proregion and mature domain each.

Two condensed multifurcating trees were constructed,

emphasizing the reliable portion of pattern branches

Characterization of cathelicidins from C. coturnix F. Feng et al.

1574 FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS

(Fig. 4). Fig. 4A reveals that there is very little differ-

ence in the proregion segment of CATH1 and CATH3;

thus, they are considered to show evolutionary ‘close-

ness’ because there has been insufficient time for many

mutations to accumulate in their proregion. For

CATH2 (fowlicidin-2, Pc-CATH2 and Cc-CATH2),

the more different proregions from CATH1 and 3 were

observed (the less homology shown) (Fig. 3B), indicat-

ing the further evolutionary distance of CATH2 from

CATH1 and 3, as well as the greater length of time

CATH2 since they shared a common ancestor. In

addition, CATH1 and 3 from C. coturnix fall into one

branch, and CATH1 and 3 from Phasianus colchicus

and chicken fowlicidins are in another branch, suggest-

ing that cathelicidins in the C. coturnix-specific cluster

arose earlier from a common ancestor than the other

two species. Unlike the highly distinct mammalian

cathelicidins resulting from repeating gene duplication

events and subsequent divergence, phylogenetic analy-

sis of the mature peptide segment revealed significant

similarity of avian cathelicidin-derived antimicrobial

peptides, as supported by bootstrap values of up to

100% (Fig. 4B). One possible explanation might be

that the much stronger activity of Aves cathelicidin

(compared with Reptilia and Mammalia) is a result of

it having undergone much less gene evolution [28].

Cc-CATH1 ATGCTGAGCTGCTGGGTGCTGGTGCTGGCGCTGCTGGGGGGGGCCTGTGCCCTCCCGGCC 60

Cc-CATH2 -------------------------------------------T---------------- 60

Cc-CATH3 ------------------------------------------------------------ 60

Cc-CATH1 CCCCTGGATTACAACCAGGCTCTGGCCCAGGCTGTGGACTCCTACAACCAACGGCCCGAG 120

Cc-CATH2 --T---AGC---CC------G---AT-------------A-------------------- 120

Cc-CATH3 --------C--------------------------------------------------- 120

Cc-CATH1 GTGCAGAATGCCTTCAGGCTGCTCAGCGCCGACCCCGAACCCGGCCCAAACGTCCAGCTC 180

Cc-CATH2 -C---------------------------------------T------GG-A-TG-T--G 180

Cc-CATH3 ------------------------------------------------------------ 180

Cc-CATH1 AGCTCCCTGCACAACCTCAACTTCACCATCATGGAGACGCGGTGCCAGGCGCGTTCGGGT 240

Cc-CATH2 -A-A-G----GGG-G-----------------------CGA----GTCC-CA-CG-AC-G 240

Cc-CATH3 ------------------------------------------------------------ 240

Cc-CATH1 GCCCAGCTTGAAAGCTGCGACTTCAAGGAGGACGGGCTCGTCAAGGACTGCGCTGCGCCC 300

Cc-CATH2 A-A-GCA-C--TGA----------------A-----GC-A-----------T-G-G---A 300

Cc-CATH3 ------------------------------------------------------------ 300

Cc-CATH1 GTGGTGCTGCAAGGCGGCCGCGCCGTGCTCGATGTCACCTGCGTGGACTCCATGGCTGAT 360

Cc-CATH2 ---ACCA-C-TGCAG-A-GCAC-T-A-A--AGCC-G-A----AGA---G--TC-T-G--- 360

Cc-CATH3 ------------------------------------------------------------ 360

Cc-CATH1 CCTGTCCGTGTCAAGCGCGTCTTGCCGCTGGT.........CATCAGGACTGTGATTGCA 411

Cc-CATH2 --C----TC---C----G....G----G-TTGGCC...................GC-T-C 397

Cc-CATH3 -------------G----T---G---------GCCGGTGGC-----AC--G---GC---G 420

Cc-CATH1 GGATACAACCTCTACCGGGCAATCAAGAGGAAGTGAgccgtccccagagctgctgtcacc 471

Cc-CATH2 T--AGA-GGTC-G--GCTT--TC-CTA---TCA-C-T-GCCG------T-G---CA-G-T 457

Cc-CATH3 --CAT----------AAA--C----G------ATGA---acg-t----------c----- 480

Cc-CATH1 actgtcccctcgctgccttccatccaataaaggtctttgctggtaaaaaaaaaaaaaaaa 531

Cc-CATH2 TTG-CTGAg-gaataaa-ggggc--gtgtg---c-accaagc-a---------------- 517

Cc-CATH3 g-------tc---a---cc---c--aataaa-c-g---ttca-gct-------------- 540

Cc-CATH1 aaaaaaaaaaaaaa 545

Cc-CATH2 -------------- 531

Cc-CATH3 --------------a 555

Fig. 1. Alignment of the cDNA sequences

of three Cc-CATHs. The stop codons (‘TGA’)

are shown in bold. Dashes represent similar

sequences. The 3¢-UTR is shown in lower-

case letters. The potential polyadentlation

signal (aataaa) is underlined. Gaps are

inserted to maximize the similarity.

F. Feng et al. Characterization of cathelicidins from C. coturnix


Cc-CATH2 expression and purification

In the Escherichia coli BL21 and pET-32a(+) plasmid

protein expression system, the deduced mature Cc-

CATH2 was expressed directly as a His-tagged fusion

protein. After induction with 1 mm isopropyl thio-b-d-galactoside (IPTG) for 4 h, a high expression level of

fusion protein was noted in E. coli BL21 (Fig. 5A).

However, the fusion protein was primarily produced as

the inclusion body (Fig. 5B). After denaturation and

His-tag affinity chromatography, the fusion protein

was renatured and examined by SDS ⁄PAGE gel

(Fig. 5C), indicating a clear and unique protein band

of 21.7 kDa, which matched well with the theoretical

mass of the fusion protein.

After formic acid cleavage for almost 24 h at 50 �C,the fusion protein was cleaved into two parts: rCc-

CATH2 (� 3.8 kDa) and carrier protein (� 16.9 kDa).

The reaction mixture was lyophilized to remove formic

acid and then the rCc-CATH2 was subjected to further

purification by RP-HPLC. The antibacterial activity of

rCc-CATH2 toward Staphylococcus aureus ATCC2592

was examined by an inhibition zone assay, and a clear

inhibition zone was observed around the spot of the

peptide, indicating that the recombinant Cc-CATH2

retained antimicrobial activity.

Antimicrobial activity of Cc-CATHs

Cc-CATH2 and Cc-CATH3 were commercially synthe-

sized by the standard solid phase synthesis method

and purified to > 95% purity. LL-37 characterized

from humans and the antibiotics, ampicillin and kana-

mycin, were used as positive controls. Essentially,

Cc-CATH2 and Cc-CATH3 showed strong and broad-

spectrum antimicrobial activities against most of the

tested microorganisms, especially a number of clinical

drug-resistant strains (Table 1). For most strains, the

minimum inhibitory concentration (MICs) are within

the range 1.3–2.5 lm, with and without the presence of

100 mm NaCl, whereas ampicillin, kanamycin and

LL-37 often did not show detectable activity in an

inhibition zone assay at dose of up to 2 mgÆmL)1. The

lowest MICs of Cc-CATH2 and 3 were detected both

for S. aureus ATCC25922, 0.3 and 0.2 lm, respec-

tively. With respect ot several Gram-positive S. aureus

clinical strains, Cc-CATH3 showed an almost ten-fold

higher activity than Cc-CATH2. However, for most of

Gram-negative bacteria tested, the result was opposite

(i.e. Cc-CATH2 was much more active than Cc-

CATH3). For example, the MIC of Cc-CATH2 to

E. coli ATCC25922 was as low as 2.5 lm, although no

detectable activity was observed for Cc-CATH3 at

2 mgÆmL)1. By contrast to LL-37 and EA-CATH1

(cathelicidin-derived antimicrobial peptides from

Equus asinus), which have weak Gram-negative bacte-

ricidal activities [32], Cc-CATH2 exerted comparable

antimicrobial activity upon most of the E. coli, with

MICs in the range 1.3–2.5 lm.

The effect of sodium upon the antimicrobial

activities of Cc-CATH2 and 3 was also examined

(Table 1). Unlike many antimicrobial peptides for

which activities are inhibited by sodium at physiologi-

cal concentrations [33–37], Cc-CATH2 and 3 showed

salt-independent activities with or without the presence

of 100 mm NaCl (Table 1), suggesting their suitability

for both local and systemic therapeutic applications.

Cytotoxicity, hemolysis of Cc-CATHs

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide method was exploited to evaluate the cytotox-

icity of Cc-CATHs toward two mammalian cell lines,

HUVEC (human umbilical vein endothelial cells) and

Raw 264.7. The results obtained revealed average IC50

values of 75 lgÆmL)1 (20.18 lm) for Cc-CATH2 and

58 lgÆmL)1 (17.16 lm) for Cc-CATH3 toward both

cell lines, which is almost ten-fold higher than their

corresponding MICs, suggesting the potential for thera-

peutic application.

Cc-CATH1 MLSCWVLVLALLGGACALPAPLDYNQALAQAVDSYNQRPEVQNAFRLLSADPEPGPNVQL 60

Cc-CATH2 --------------V-------S-P---I----T------A---------------GID- 60

Cc-CATH3 ------------------------------------------------------------ 60

Cc-CATH1 SSLHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMAD 120

Cc-CATH2 NT-RE--------E-VPSARTRIDD-----N-AI---SG--TILQDAPEISLN-R-ASS- 120

Cc-CATH3 ------------------------------------------------------------ 120

Cc-CATH1 PVRVKRVLPLV...IRTVIAGYNLYRAIKRK 148

Cc-CATH2 --L-Q-GRFGRFLKKVRRFIPKVIIA-QIGSRFG 154

Cc-CATH3 --RVR-FW---PVA-N--A--I---K--R-- 151

Fig. 2. Alignment of the predicted precursor

amino acid sequences of the Cc-CATHs.

Gaps are inserted to optimize the alignment.

Identical residues are indicated by dashes.



A possible limitation to the clinical application of

antimicrobial peptides as antibiotics is their potential to

cause injury to mammalian cell membranes. In the

present study, the hemolytic activities of Cc-CATHs

were also examined using freshly prepared human

erythrocytes. As shown in Table 2, Cc-CATH2 and 3

both displayed negligible hemolytic activities, lysing only

3.6% and 4.1% of erythrocytes at concentrations up to

26.9 lm (100 lgÆmL)1) and 29.6 lm (100 lgÆmL)1),

respectively. The hemolysis concentrations are much

62Pc-CATH1 62Pc-CATH2 62Pc-CATH3 62Cc-CATH1 62Cc-CATH2 62Cc-CATH3 62Fowlicidin1 62Fowlicidin2 62Fowlicidin3 72Ea CATH1 72Ec CATH1 72Ec CATH2 72Ec CATH3 73Hs LL37 72Ss PR39 72Bt CATHL1 73Oa SMAP29 72Ch BAC5 73Cp CAP11 70Mm CRAMP 72Oc CAP18 72Clf K9CATH 68Bf cath

........MLSCWVLVLALLGGACALPAP...LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLGS

........MLSCWVLVLALLGGVCALPAP...LSYPQALTQAVDSYNQRPELQ.NAFRLLSADPEPGPG.VDLST

........MLSCWVLVLALLGGACALPAP...LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLGS

........MLSCWVLVLALLGGACALPAP...LDYNQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS

........MLSCWVLVLALLGGVCALPAP...LSYPQALIQAVDTYNQRPEAQ.NAFRLLSADPEPGPG.IDLNT

........MLSCWVLVLALLGGACALPAP...LDYNQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS

........MLSCWVLLLALLGGACALPAP...LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS

........MLSCWVLLLALLGGVCALPAP...LSYPQALIQAVDSYNQRPEVQ.NAFRLLSADPEPGPG.VDLST

........MLSCWVLLLALLGGACALPAP...LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSSMETQRDSCSLGWWSLLLLLLGLMIPLATT.QALSYKEAVLRAVDGLNQWSSDE.NLYRLLELDPLPKGD.EAPDTMETQRNTRCLGRWSPLLLLLGLVIPPATT.QALSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKGD.KDSDTMETQRDSCSLGRWSLLLLLLGLVIPLATT.QTLSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKED.EDPDTMETQRNTRCLGRWSPLLLLLGLVIPPATT.QALSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKGD.KDSDTMKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDA.NLYRLLDLDPRPTMD.GDPDTMETQRASLCLGRWSLWLLLLGLVVPSAST.QALSYREAVLRAVDRLNEQSSEA.NLYRLLELDQPPKAD.EDPGTMETPRASLSLGRWSLWLLLLGLALPSASA.QALSYREAVLRAVDQLNEQSSEP.NIYRLLELDQPP.QDDEDPDSMETQRASLSLGRRSLWLLLLGLVLASARA.QALSYREAVLRAVDQLNEKSSEA.NLYRLLELDPPPKQDDENSNIMETQGASLSLGRWSLWLLLLGLVVPLASA.QALSYREAVLRAVGQLNERSSEA.NLYRLLELDPAPNDE.VDPGTMGTPRDAASGGPRLLLPLLLLLLLTPATA.WVLSYQQAVQRAVDGINKNLADNENLFRLLSLDTQPPGD.NDPYSMQFQRDVPSLWLWRSLSLLLLLGL..GFS.QTPSYRDAVLRAVDDFNQQSLDT.NLYRLLDLDPEPQGD.EDPDTMETHKHGPSLAWWSLLLLLLGLLMPPAIA.QDLTYREAVLRAVDAFNQQSSEA.NLYRLLSMDPQQLED.AKPYTMETQKDSPSLGRWSLLLLLLGLVITPAAS.RALSYREAVLRAVNGFNQRSSEE.NLYRLLQLNSQPKGD.EDPNI....MEGFFWKTLLVVGALAIAGTSSLPH.KPLIYEEAVDLAVSIYNSKSGEDS.LYRLLEAVSPPKWD.PLSES

LLLLLLLLLLLLLLLLLLLLLLL

YYYYYYYYYYYYYYYYYYYYYYY

AAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAA

NNNNNNNNNNNNNNNNNNNNNNN

RRRRRRRRRRRRRRRRRRRRRRR




LHNLNFTIIETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRATFDVTCVESVADPV...............LRTLNFTIMETECVPRAQTPIDDCDFKENGVIRDCSGPVTILQDTPEINLRCRDASSDPV...............LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRATFDVTCVDSMADPV...............LHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV...............LRELNFTIMETECVPSARTRIDDCDFKENGAIKDCSGPVTILQDAPEISLNCRDASSDPV...............LHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV...............LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV...............LRALNFTIMETECTPSARLPVDDCDFKENGVIRDCSGPVSVLQDTPEINLRCRDASSDPV...............LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV...............PKPVSFTVKETVCPRTTQQPLEQCDFKENGLVKQCVGTVILDPVKASVDIGCDEPQRV.................PKPVSFMVKETVCPRIMKQTPEQCDFKENGLVKQCVGTVILGPVKDHFDVSCGEPQRV.................PKPVSFTVKETVCPRTTQQPLEECDFKENGLVKQCVGTVVLDPAKDYFDISCDKPQPI.................PKPVSFMVKETVCPRIMKQTPEQCDFKENGLVKQCVGTVILDPVKDYFDASCDEPQRV.................PKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFA...............PKPVSFTVKETVCPRPTQRPPELCDFKENGRVKQCVGTVTLNPSNDPLDISCNEIQSV.................PKRVSFRVKETVCSRTTQQPPEQCDFKENGLLKRCEGTVTLDQVRGNFDITCNNHQSIRITKQPWAPPQAA....PKPVSFRVKETVCPRTSQQPAEQCDFKENGLLKECVGTVTLDQVGNNFDITCAEPQSV.................RKPVSFTVKETVCPRTTQQPPEECDFKENGLVKQCVGTVTLDPSNDQFDINCNELQSV.................PKPVSFTIKETVCTKMLQRPLEQCDFKENGLVQRCTGTVTLDSAFNVSSLSCLGGRRF.................PKSVRFRVKETVCGKAERQLPEQCAFKEQGVVKQCMGAVTLNPAADSFDISCNEPGAQPFRFKKISRLA......PQPVSFTVKETECPRTTWKLPEQCDFKEDGLVKRCVGTVTRYQAWDSFDIRCNRAQESPEPT.............PKPVSFTVKETVCPKTTQQPLEQCGFKDNGLVKQCEGTVILDEDTGYFDLNCDSILQVKKID.............NQELNFTMKETVCLVAEERSLEECDFQEDGVVMGCTGYYFFGESPPVVVLTCKPVGEEGEQKQEEGNEEEKEVEE

FFFFFFFFFFFFFFFFFFFFFFF

EEEEEEEEEEEEEEEEEEEEEEE

TTTTTTTTTTTTTTTTTTTTTTT

CCCCCCCCCCCCCCCCCCCCCCC


FFFFFFFFFFFFFFFFFFFFFFF

GGGGGGGGGGGGGGGGGGGGGGG




....................RIKRFWPVVIRTVVAGYNLYRAIKKK

....................LVQRGRFGRFLSKIRRFRPKFTITIQGSGRFG

....................RIKRFWPLVPVAINTVAAGINLYKAIKRK

....................RVKRVLPLVIRTVIAGYNLYRAIKRK

....................LVQRGRFGRFLKKVRRFIPKVIIAAQIGSRFG

....................RVRRFWPLVPVAINTVAAGINLYKAIRRK

....................RVKRVWPLVIRTVIAGYNLYRAIKKK

....................LVQRGRFGRFLRKIRRFRPKVTITIQGSARFG

....................RVKRFWPLVPVAINTVAAGINLYKAIRRK

....................KRRGSVTTRYQFLMIHLLRPKKLFA

....................KRFGRLAKSFLRMRILLPRRKILLAS

....................KRRHWFPLSFQEFLEQLRRFRDQLPFP

....................KRFHSVGSLIQRHQQMIRDKSEATRHGIRIITRPKLLLAS

....................LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

....................RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR

....................RLCRIVVIRVCR

....................RGLRRLGRKIAHGVKKYGPTVLRIIRIAG

....................RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFPGRR

....................RRMVGLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCR

....................GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE

....................GLRKRLRKFRNKIKEKLKKIGQKIQGFVPKLAPRTDY

....................RLKELITTGGQKIGEKIRRIGQRIKDFFKNLQPREEKSEEQEEDEKDQPRRV......KRFKKFFRKLKKSVKKRAKEFFKKPRVIGVSIPF

A

Fig. 3. (A) Multiple sequence alignment of Cc-CATHs with classic cathelicidins from different species. The conserved amino acid residues in

cathelin domain are shaded, including the typical four conserved cysteine residues. Each mature cathelicidin is aligned in the third line. Pc,

P. colchicus (ring necked pheasant); Fowlicidin (chicken); Hs, Homo sapiens (human); Ss, Sus scrofa (pig); Bt, Bos taurus (cattle); Oa, Ovis

aries (sheep); Ch, Capra hircus (goat); Cp, Cavia porcellus (guinea pig); Ec, Equus caballus (horse); Mm, Mus musculus (mouse); Oc, Oryctol-

agus cuniculus (rabbit); Clf, Canis lupus familiars (dog); Bf, Bungarus fasciatus (snake). Dots are inserted to maximize the similarity. (B) Align-

ment of Cc-CATHs with avian cathelicidins. Each mature cathelicidin is boxed.



higher than the corresponding MICs, except for two of

the S. aureus clinical isolated strains (Table 1), suggest-

ing the considerable selectivity of Cc-CATH2 and 3 for

microorganisms over mammalian cells in vitro.

Discussion

The emergence of widespread antibiotic resistance in

numerous commonly encountered bacteria requires the

discovery of new bactericidal agents with therapeutic

potential. Currently, a new superbug is being reported

that is resistant to even the most powerful antibiotics,

and has produced dangerous infections in countries

such as the USA, Canada, Australia and the Nether-

lands [38]. The bacteria synthesizes an enzyme called

NDM-1 that can exist inside different bacteria, such as

E. coli, making them resistant to one of the most pow-

erful groups of antibiotics (i.e. carbapenems). There-

fore, tight surveillance and new drugs are needed to

manage this threat. The cathelicidin family of endoge-

nous antimicrobial peptides serves a critical role in

mammalian innate immune defense against invasive

bacterial infection [39]. The cathelicidin-derived antimi-

crobial peptides have recently received attention

because of their much stronger bactericidal activities

compared to chemical drugs, as well as their unique

killing mechanism, as a result of which drug resistance

is difficult to develop. They kill microorganisms by cre-

ating pores or holes in pathogen membranes, unlike

the conventional b-lactam antibiotics, which kill most

bacteria by inhibiting the synthesis of one of their cell

wall layers [40,41]. Cathelicidins can kill both Gram-

positive and -negative bacteria, enveloped viruses

including HIV, and fungi including Candida and Cryp-

tococcus [3]. As antibiotics, cathelicidins are also effec-

tive against resistant staphylococcus, enterococcus and

pseudomonas in animal models [34,42,43]. They are

also found to bind lipopolysaccharide or recruit the

immune system, and to inhibit reactive oxygen species

created by neutrophils, thus mitigating excess tissue

damage [44–46].

In the present study, three cathelicidins were identi-

fied from a C. coturnix cDNA library. The cDNAs of

Cc-CATHs demonstrate the same conserved cathelici-

din family gene organization, including the signal

B

Fig. 3. (Continued).

A Fowlicidin 1

Fowlicidin 3

PC-CATH1

PC-CATH3

CC-CATH-1

CC-CATH-3

Fowlicidin 2

PC-CATH2

CC-CATH-2

98

95

94

70

100

B CC-CATH-3

Fowlicidin 3

PC-CATH3

CC-CATH-1

Fowlicidin 1

PC-CATH1

CC-CATH-2

PC-CATH2

Fowlicidin 2

84

81

58

53

79

100

Fig. 4. Phylogenetic analyses of Cc-CATHs and avian cathelicidins

on the basis of the proregion (A) and mature domain (B). The phylo-

genetic dendrogram was constructed by the Neighbor-joining

method based on the proportion difference of aligned amino acid

sites of the sequence. Only branches supported by a bootstrap

value of at least 50% (expressed as percentage of 1000 bootstrap

samples supporting the branch) are shown at the branching points.



peptide, the cathelin domain, and the deduced mature

antimicrobial peptide of 26, 32 and 29 amino acid resi-

dues, respectively. Moreover, the four highly conserved

cysteines were also maintained in the pro-region

sequences. Cc-CATH1-3 is markedly conserved with

chicken fowlicidin-1–3. However, the data obtained

from antimicrobial testing indicated that Cc-CATH2

was not as strongly active as its pair fowlicidin-2, and

Cc-CATH3 was also less active compared to fowlici-

din-1 and -2 [16]. The MICs of fowlicidin-1 and -2 are

in the range 0.4–2.0 lm for most strains [16]. Another

cathelicidin-derived peptide, Pc-CATH1 (pairs with

fowlicidin1 and Cc-CATH1), which was identified

from P. colchicus in a previous study [28], also pos-

sesses potent antimicrobial activity, with most MICs

in the range 0.09–2.95 lm. To explain the different

bactericidal performances of these peptides that have

great sequence similarity, their secondary structures

were predicted online using gor iv (http://npsa-pbil.

ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html).

The results obtained demonstrated that the a-helicalcontent for the ‘strong group’, including fowlicidin 1-2

and Pc-CATH1, is 38.46%, 38.71% and 38.46%,

respectively. For the ‘weak group’, Cc-CATH2 and 3,

the a-helical content is 62.50% and 65.52%, respec-

tively, which is almost two-fold higher than the ‘strong

group’. Although the a-helical structure is considered

to be responsible for the formation of pores in the

membranes of target microorganisms [47], the results

of the present study indicate that the percentage of the

a-helix must be within an optimal range for the pep-

tide to achieve its best activity.

Although the antimicrobial activities of Cc-CATHs

are not as potent as those of Pc-CATH1 and fowlici-

din-1 and -2, the hemolytic activities of Cc-CATHs are

significantly lower. The considerable reduction of cyto-

toxic activity, as well as potent and broad-spectrum

antimicrobial activity, even against clinical drug-resis-

tant strains, offers a marked improvement in terms of

the application of Cc-CATHs for the treatment of bac-

terial and fungal infections.

Materials and methods

Collection of tissues

Two adult female quails were captured from Zhengding,

Hebei Province of China. One quail was killed and the

spleen was dissected immediately and frozen in liquid nitro-

gen until use.

Total RNA extraction and SMART cDNA

synthesis

Total RNA was extracted from the spleen of quail using

RNeasy Mini Kit (Qiagen, Hildenberg, Germany) in accor-

dance with the manufacturer’s instructions. cDNA synthesis

was carried out by a PCR-based method using a Creator�

kDa

21.7 kDa

kDa 1 2 3 4 0 5 6 7 80 1 2 3 4

10kDa10080605040

30

20

12

2 3 4

170A B

C

130100

70

55

40

35

25

15

170130100

70

55

40

35

25

15

Fig. 5. (A) Expression and purification of

Cc-CATH2 fusion protein (indicted by an

arrow) followed by SDS ⁄ PAGE (15%). Lane

1, the whole lysate without IPTG; lanes 2–4,

the whole lysate with 1 mM IPTG for 4 h;

lane 0, protein standards (kDa). (B) The

results of SDS ⁄ PAGE (15%) for supernatant

and precipitation at the same time. Lanes

1–3, precipitation with IPTG; lane 4, precipi-

tation without IPTG; lane 5, supernatant

without IPTG; lanes 6–8, supernatant with

IPTG; lane 0, protein standards. (C) Protein

bands after affinity chromatography and

renaturing process. Lanes 1 and 2, protein

bands after separation by affinity column;

lanes 3 and 4, protein bands after renaturing

process: lane 0, protein standards.



Table 1. Antimicrobial activity of Cc-CATHs. MIC, minimal inhibitory concentration (these concentrations represent the mean values of three

independent experiments performed in duplicate); Amp, ampicillin; Kana, kanamycin; ND, no detectable activity in inhibition zone assay at a

dose of 2 mgÆmL)1; IS, clinically isolated strain; Dra, drug resistance for ceftazidime, cefoperazone and aztreonam; DRb, drug resistance for

compound sulfamethoxazole, erythromycin, ciprofloxacin and penicillin.

Microorganism

MIC (lM)

Cc-CATH2

(0 mM NaCl)

Cc-CATH2

(100 mM NaCl)

Cc-CATH3

(0 mM NaCl)

Cc-CATH3

(100 mM NaCl)

LL-37

(0 mM NaCl) Amp Kana

Gram-positive

Staphylococcus aureus

ATCC2592

0.3 0.3 0.2 0.4 1.0 0.79 8.05

S. aureus 1.3 1.3 0.7 0.7 4.2 6.31 128.74

S. aureus (IS 1303) 20.2 20.2 2.8 2.8 ND 100.97 ND

S. aureus (IS 1307) > 26.9 > 26.9 2.8 2.8 > 22.3 6.31 ND

S. aureus (IS 1348) 20.2 20.2 2.8 2.8 > 22.3 50.48 ND

S. aureus (IS 1349) 20.2 20.2 2.8 2.8 > 22.3 100.97 ND

S. aureus (IS 1350) 20.2 20.2 2.8 2.8 ND 100.97 ND

Staphylococcus epidermidis 2.5 2.5 ND ND ND 201.94 4.02

Staphylococcus haemolyticus

(IS 2401, DRa)

2.5 2.5 0.7 0.7 ND 1.58 16.09

Nocardia asteroids 1.3 1.3 0.7 0.7 4.2 3.16 128.74

Enterococcus faecalis (IS 981) 1.3 1.3 5.6 11.1 > 22.3 201.94 ND

Enterococcus faecium (IS 1299) 2.5 2.5 1.4 1.4 4.2 ND ND

Propionibacterium acnes

ATCC 11827

1.3 2.5 1.4 1.4 > 22.3 3.16 4.02

Gram-negative

Klebsiella oxytoca 2.5 2.5 22.2 22.2 ND ND ND

Aeromonas sobria 1.3 1.3 1.4 2.8 4.2 ND ND

Acinetobacter baumannii

(IS 2178, DRb)

1.3 1.3 1.4 1.4 ND 100.97 4.02

A. baumannii (IS 2373) 2.5 1.3 1.4 2.8 ND ND ND

Stenotrophomonas maltophilia 1.3 1.3 1.4 2.8 4.2 ND ND

S. maltophilia (IS 1404) 0.6 0.6 5.6 5.6 > 22.3 ND ND

Pseudomonas aeruginosa

ATCC 27853

10.1 10.1 5.6 11.1 ND ND ND

P. aeruginosa (IS 1411) 10.1 10.1 5.6 5.6 > 22.3 ND ND

P. aeruginosa (IS 1412) 10.1 10.1 5.6 11.1 ND ND ND

P. aeruginosa (IS 1413) 10.1 10.1 5.6 5.6 ND > 269.25 128.74

Escherichia coli ATCC 25922 2.5 2.5 ND ND ND 25.24 16.09

E. coli 5.1 2.5 ND ND ND 201.94 4.02

E. coli (IS 1334) 2.5 1.3 11.1 11.1 ND ND 32.18

E. coli (IS 1335) 1.3 2.5 5.6 5.6 ND ND ND

E. coli (IS 1342) 1.3 1.3 2.8 2.8 ND ND 32.18

E. coli (IS 1375) 2.5 2.5 11.1 11.1 > 22.3 50.48 4.02

Serratia marcescens (IS 1379) ND ND ND ND ND ND ND

Klebsiella pneumoniae (IS 1368) 1.3 1.3 ND ND 4.2 ND 64.37

K. pneumoniae (IS 1372) 2.5 2.5 ND ND ND ND ND

K. pneumoniae (IS 1373) 2.5 2.5 ND ND ND ND 4.02

K. pneumoniae (IS 1400) 5.1 5.1 22.2 22.2 ND ND ND

Proteus vulgaris 10.1 10.1 > 29.6 > 29.6 ND 3.16 8.05

Proteus mirabilis 5.1 5.1 1.4 1.4 ND 6.31 8.05

Salmonella typhi (IS 1408) 5.1 5.1 ND ND ND ND 32.18

Fungi

Candida albicans ATCC 2002 1.3 1.3 0.7 0.7 2.1 1.58 2.01

Candida glabrata (IS 0902) 10.1 10.1 5.6 5.6 ND ND ND

Slime mold 0.6 1.3 0.7 0.7 4.2 6.31 128.74



SMART� cDNA library construction kit (Clontech, Palo

Alto, CA, USA). First-strand cDNA was synthesized by

SMART� IV oligonucleotide primer 5¢-AAGCAGTGG-

TATCAACGCAGAGTGGCCATTACGGCCGGG-3¢ and

CDS III ⁄ 3¢ PCR primer 5¢-ATTCTAGAGGCCGAGGC

GGCCGACATGT (30)N–1N-3¢ (N = A, G, C or T; N–1

= A, G or C); the reverse transcriptase used was Power-

Script Reverse Transcriptase, as supplied with the kit.

Second-strand cDNA was amplified by 5¢ PCR primer

5¢-AAGCAGTGGTATCAACGCAGAGT-3¢ and CDS

III ⁄ 3¢ PCR primer, using Advantage DNA Polymerase

from Clontech.

Screening of cathelicidin-encoding cDNAs and

phylogenetic tree construction

On the basis of the conserved signal domain of previously

characterized chicken fowlicidin cDNAs [21], two sense

primers P1 (5¢-AGGATGCTGAGCTGCTGGGT-3¢) and

P2 (5¢-ATGCTGAGCTGCTGGGTGCT-3¢) were designed

from 5¢-UTR and a highly conserved domain encoding the

signal peptide of fowlicidins, and coupled with CDS III ⁄ 3¢PCR primer. The half nested PCR conditions consisted of

two parts. The first part comprised: 94 �C for 1 min; 20

cycles of 94 �C for 20 s, 60 �C for 30 s, 72 �C for 60 s; fol-

lowed by a final extension at 72 �C for 5 min. The second

part comprised: 94 �C for 3 min; 25 cycles of 94 �C for

20 s, 58 �C for 30 s, 72 �C for 60 s; followed by a final

extension at 72 �C for 10 min. The PCR product was puri-

fied by gel electrophoresis and cloned into pGEM-T vector

(Promega, Madison, WI, USA). DNA sequencing was per-

formed using an ABI PRISM 377 (Applied Biosystems,

Foster City, CA, USA).

In total, nine avian cathelicidin sequences were obtained

from the protein database at the National Center for Bio-

technology Information. These were the fowlicidins [16],

Pc-CATHs [28] and Cc-CATHs from the present study.

Multisequence alignments were constructed using clu-

stalw, version 1.8 (http://www.ebi.ac.uk/clustalw/), based

on the proregion and mature domain. The phylogenetic

trees were constructed using the Neighbor-joining method

(mega, version 4.0; www.megasoftware.net), by calculating

the proportion of amino acid differences (p-distance)

among all sequences. A total of 1000 bootstrap replicates

were used to test the reliability of each branch. The num-

bers on the branches indicate the percentage of 1000 boot-

strap samples supporting the branch.

Expression vector construction, protein

expression and purification

Host strain E. coli BL21 and pET-32a(+) plasmid (Nov-

agen, Darmstadt, Germany) was utilized for Cc-CATH2

expression. The method was carried out in accordance with

the manufacturer’s instructions and as described previously

by Li et al. [48].

The two restriction sites for KpnI and HindIII and the

formic acid cleavage site (AspPro) upstream of the deduced

mature Cc-CATH2 coding sequence were utilized in the

peptide expression. A DNA fragment encoding the gene for

Cc-CATH2 was amplified by PCR from the plasmids

described above. The first forward primer was 5¢-AC-

CGACCCGCTCGTCCAGCG-3¢ and the first reverse pri-

mer was 5¢-CTTCTAGCCAAAGCGTGAGCCGATC-3¢.PCR was performed by running 30 cycles with a tempera-

ture profile of 30 s at 94 �C, 30 s at 64 �C and 10 s at

72 �C followed by a final extension at 72 �C for 10 min.

The second forward primer was 5¢-CGGGGTACC

GACCCGCTCGT-3¢ and the second reverse primer was 5¢-CCCAAGCTTCTAGCCAAAGCGTG-3¢. PCR comprised:

30 cycles of 30 s at 94 �C, 30 s at 64 �C and 10 s at 72 �C,followed by a final extension at 72 �C for 10 min. The puri-

fied PCR product was digested with KpnI and HindIII, and

ligated into the pET-32a(+) plasmid at the corresponding

restriction sites. The resultant recombinant vector is

referred to as Cc-CATH2 ⁄ pET-32a(+). The Cc-CATH2 ⁄pET-32a(+) construct was transformed into the E. coli

strain BL21 for protein expression. The fusion protein

expression was initiated by adding IPTG.

After lysis by sonication, the whole cell lysate was then

centrifuged at 3914 g for 15 min, and then the supernatant

and precipitation were both resolved by SDS ⁄PAGE. After

centrifugation, the fusion protein was found primarily in

the precipitation. The inclusion body was collected, washed

and resolved by denaturant solution. The solution was col-

lected and purified with a His-tag affinity column. After re-

natured in gradient, the Cc-CATH2-containing fusion

protein was cleaved in 50% formic acid (v ⁄ v) at 50 �C for

24 h. After lyophilization, the solution was subject to

HPLC (Hypersil BDS C18, Elite, Dalian, China;

30 · 0.46 cm). The peptide was eluted by a mixture of sol-

vents of acetonitrile ⁄H2O ⁄ 0.1% trifluoroacetic acid at a

flow rate of 1 mLÆmin)1 using a linear gradient of increas-

ing acetonitrile. Fractions corresponding to the major peak

were collected and lyophilized. Subsequently, the anti-

bacterial activity of expressed Cc-CATH2 with respect to

S. aureus ATCC2592 was examined.

Peptide synthesis

The deduced cathelicidin-derived mature peptides, LL-37,

Cc-CATH2 and 3 were synthesized by the peptide synthe-

sizer GL Biochem (Shanghai) Ltd. (Shanghai, China) and

Table 2. Hemolysis assay of Cc-CATHs.

Hemolytic activity

Concentration (lgÆmL)1) 100 50 20 10

Cc-CATH2 (%) 3.6 0.0 0.0 0.0

Cc-CATH3 (%) 4.1 1.3 0.0 0.0



analyzed by HPLC and MALDI-TOF MS to confirm that

the purity was higher than 98%. All peptides were dissolved

in water and used for activity examination, as described

below.

Antimicrobial assay

To examine the antibacterial spectrum of Cc-CATHs, a

modified broth microdilution assay was used as described

in a previous study [49]. The microorganisms evaluated

included standard and clinically isolated drug resistance

bacterial and fungal strains (Table 1). Briefly, bacteria were

subcultured to the midlogarithmic phase at 37 �C and sus-

pended to 5 · 105 colony-forming unitsÆmL)1 in Mueller–

Hinton (MH) broth with and without 100 mm of NaCl.

The peptides in the presence and absence of 100 mm NaCl

were subjected to serial dilutions in MH broth, and then

50 lL of the diluted samples was dispensed into a 96-well

microtiter plate and mixed with 50 lL of bacteria or yeast

inoculums in MH. Human cathelicidin LL-37 (without

NaCl), ampicillin and kanamycin was used as a positive

control. The microtiter plate was incubated at 37 �C for

18 h for bacteria and 48 h for fungus, and A595 was mea-

sured. MIC was defined as the lowest concentration of pep-

tide that completely inhibits the growth of the microbe as

determined by visual inspection and spectrophotometric

examination.

Cytotoxicity assay

HUVEC and Raw 264.7 murine macrophage cells were

used to examine the in vitro cytotoxicity of Cc-CATHs. The

cells were cultured in DMEM (Gibco, Gaithersburg, MD,

USA) supplemented with 10% fetal bovine serum,

100 UÆmL)1 of penicillin and 100 UÆmL)1 of streptomycin

in a humidified 5% CO2 atmosphere at 37 �C. Cells

(2 · 104 per well) were seeded in 96-well plates and cultured

overnight until they adhered to the plate. Various concen-

trations of Cc-CATHs dissolved in the corresponding cul-

ture medium were added to the wells and the plates were

incubated at 37 �C for 48 h. Cytotoxicity of Cc-CATHs

was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-

nyltetrazolium bromide method [50]. IC50 was defined as

the concentration of Cc-CATHs at which A490 was reduced

by 50%.

Hemolysis

Hemolysis assays were conducted as described previously

[51]. Cc-CATHs of four different concentrations were incu-

bated with washed human erythrocytes at 37 �C for 30 min

and centrifuged at 652 g for 5 min and A540 of the superna-

tant was measured. 1% v ⁄ v Triton X-100 was used to

determine maximal hemolysis. The experiment was repeated

three times.

Acknowledgements

We thank the editor and the anonymous reviewers for

their helpful comments on the manuscript. This work

was supported by grants from the Chinese National

Natural Science Foundation (30900240 and 41076098).

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