Gene cloning, expression and characterization of avian cathelicidin orthologs, Cc-CATHs, from Coturnix coturnix Feifei Feng 1,2, *, Chen Chen 3, *, Wenjuan Zhu 2 , Weiyu He 1 , Huijuan Guang 2 , Zheng Li 2 , Duo Wang 1 , Jingze Liu 1 , Ming Chen 5 , Yipeng Wang 4 and Haining Yu 1,2 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: joannyu@live.cn; wyp010@163.com *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 l M for most strains with or without the presence of 100 m M NaCl. Cc-CATH2 and 3 showed considerable reduction of cytotoxic activity compared to other avian cathelicidins, with average IC 50 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 actgtcccctcgctgccttccatccaataaa ggtctttgctggtaaaaaaaaaaaaaaaa 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 C c- CATH 1 aa a a aa a a aa aa a a 5 45 C c -C AT H 2 - - - - 5 31 C c -C AT H 3 - - - - a 55 5 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 FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 1575 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 2mgÆmL )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 IC 50 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. Characterization of cathelicidins from C. coturnix F. Feng et al. 1576 FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 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.VQLSS METQRDSCSLGWWSLLLLLLGLMIPLATT.QALSYKEAVLRAVDGLNQWSSDE.NLYRLLELDPLPKGD.EAPDT METQRNTRCLGRWSPLLLLLGLVIPPATT.QALSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKGD.KDSDT METQRDSCSLGRWSLLLLLLGLVIPLATT.QTLSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKED.EDPDT METQRNTRCLGRWSPLLLLLGLVIPPATT.QALSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKGD.KDSDT MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDA.NLYRLLDLDPRPTMD.GDPDT METQRASLCLGRWSLWLLLLGLVVPSAST.QALSYREAVLRAVDRLNEQSSEA.NLYRLLELDQPPKAD.EDPGT METPRASLSLGRWSLWLLLLGLALPSASA.QALSYREAVLRAVDQLNEQSSEP.NIYRLLELDQPP.QDDEDPDS METQRASLSLGRRSLWLLLLGLVLASARA.QALSYREAVLRAVDQLNEKSSEA.NLYRLLELDPPPKQDDENSNI METQGASLSLGRWSLWLLLLGLVVPLASA.QALSYREAVLRAVGQLNERSSEA.NLYRLLELDPAPNDE.VDPGT MGTPRDAASGGPRLLLPLLLLLLLTPATA.WVLSYQQAVQRAVDGINKNLADNENLFRLLSLDTQPPGD.NDPYS MQFQRDVPSLWLWRSLSLLLLLGL GFS.QTPSYRDAVLRAVDDFNQQSLDT.NLYRLLDLDPEPQGD.EDPDT METHKHGPSLAWWSLLLLLLGLLMPPAIA.QDLTYREAVLRAVDAFNQQSSEA.NLYRLLSMDPQQLED.AKPYT METQKDSPSLGRWSLLLLLLGLVITPAAS.RALSYREAVLRAVNGFNQRSSEE.NLYRLLQLNSQPKGD.EDPNI MEGFFWKTLLVVGALAIAGTSSLPH.KPLIYEEAVDLAVSIYNSKSGEDS.LYRLLEAVSPPKWD.PLSES L L L L L L L L L L L L L L L L L L L L L L L Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A N N N N N N N N N N N N N N N N N N N N N N N R R R R R R R R R R R R R R R R R R R R R R R L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L 122Pc-CATH1 122Pc-CATH2 122Pc-CATH3 122Cc-CATH1 122Cc-CATH2 122Cc-CATH3 122Fowlicidin1 122Fowlicidin2 122Fowlicidin3 130Ea CATH1 130Ec CATH1 130Ec CATH2 130Ec CATH3 133Hs LL37 130Ss PR39 143Bt CATHL1 131Oa SMAP29 130Ch BAC5 131Cp CAP11 139Mm CRAMP 134Oc CAP18 134Clf K9CATH 143Bf cath LHNLNFTIIETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRATFDVTCVESVADPV LRTLNFTIMETECVPRAQTPIDDCDFKENGVIRDCSGPVTILQDTPEINLRCRDASSDPV LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRATFDVTCVDSMADPV LHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV LRELNFTIMETECVPSARTRIDDCDFKENGAIKDCSGPVTILQDAPEISLNCRDASSDPV LHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV LRALNFTIMETECTPSARLPVDDCDFKENGVIRDCSGPVSVLQDTPEINLRCRDASSDPV LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV PKPVSFTVKETVCPRTTQQPLEQCDFKENGLVKQCVGTVILDPVKASVDIGCDEPQRV PKPVSFMVKETVCPRIMKQTPEQCDFKENGLVKQCVGTVILGPVKDHFDVSCGEPQRV PKPVSFTVKETVCPRTTQQPLEECDFKENGLVKQCVGTVVLDPAKDYFDISCDKPQPI PKPVSFMVKETVCPRIMKQTPEQCDFKENGLVKQCVGTVILDPVKDYFDASCDEPQRV PKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFA PKPVSFTVKETVCPRPTQRPPELCDFKENGRVKQCVGTVTLNPSNDPLDISCNEIQSV PKRVSFRVKETVCSRTTQQPPEQCDFKENGLLKRCEGTVTLDQVRGNFDITCNNHQSIRITKQPWAPPQAA PKPVSFRVKETVCPRTSQQPAEQCDFKENGLLKECVGTVTLDQVGNNFDITCAEPQSV RKPVSFTVKETVCPRTTQQPPEECDFKENGLVKQCVGTVTLDPSNDQFDINCNELQSV PKPVSFTIKETVCTKMLQRPLEQCDFKENGLVQRCTGTVTLDSAFNVSSLSCLGGRRF PKSVRFRVKETVCGKAERQLPEQCAFKEQGVVKQCMGAVTLNPAADSFDISCNEPGAQPFRFKKISRLA PQPVSFTVKETECPRTTWKLPEQCDFKEDGLVKRCVGTVTRYQAWDSFDIRCNRAQESPEPT PKPVSFTVKETVCPKTTQQPLEQCGFKDNGLVKQCEGTVILDEDTGYFDLNCDSILQVKKID NQELNFTMKETVCLVAEERSLEECDFQEDGVVMGCTGYYFFGESPPVVVLTCKPVGEEGEQKQEEGNEEEKEVEE F F F F F F F F F F F F F F F F F F F F F F F E E E E E E E E E E E E E E E E E E E E E E E T T T T T T T T T T T T T T T T T T T T T T T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C F F F F F F F F F F F F F F F F F F F F F F F G G G G G G G G G G G G G G G G G G G G G G G C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 148Pc-CATH1 154Pc-CATH2 151Pc-CATH3 148Cc-CATH1 154Cc-CATH2 151Cc-CATH3 148Fowlicidin1 154Fowlicidin2 151Fowlicidin3 155Ea CATH1 156Ec CATH1 157Ec CATH2 170Ec CATH3 170Hs LL37 172Ss PR39 155Bt CATHL1 160Oa SMAP29 176Ch BAC5 177Cp CAP11 172Mm CRAMP 171Oc CAP18 172Clf K9CATH 191Bf cath RIKRFWPVVIRTVVAGYNLYRAIKKK LVQRGRFGRFLSKIRRFRPKFTITIQGSGRFG RIKRFWPLVPVAINTVAAGINLYKAIKRK RVKRVLPLVIRTVIAGYNLYRAIKRK LVQRGRFGRFLKKVRRFIPKVIIAAQIGSRFG RVRRFWPLVPVAINTVAAGINLYKAIRRK RVKRVWPLVIRTVIAGYNLYRAIKKK LVQRGRFGRFLRKIRRFRPKVTITIQGSARFG RVKRFWPLVPVAINTVAAGINLYKAIRRK KRRGSVTTRYQFLMIHLLRPKKLFA KRFGRLAKSFLRMRILLPRRKILLAS KRRHWFPLSFQEFLEQLRRFRDQLPFP KRFHSVGSLIQRHQQMIRDKSEATRHGIRIITRPKLLLAS LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR RLCRIVVIRVCR RGLRRLGRKIAHGVKKYGPTVLRIIRIAG RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFPGRR RRMVGLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCR GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE GLRKRLRKFRNKIKEKLKKIGQKIQGFVPKLAPRTDY RLKELITTGGQKIGEKIRRIGQRIKDFFKNLQPREEKS EEQEEDEKDQPRRV 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. F. Feng et al. Characterization of cathelicidins from C. coturnix FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 1577 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. Characterization of cathelicidins from C. coturnix F. Feng et al. 1578 FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 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-helical content 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 CreatorÔ kDa 21.7 kDa kDa 1 2 3 4 0 5 6 7 8 01 2 3 4 10kDa 100 80 60 50 40 30 20 12 234 170 A B C 130 100 70 55 40 35 25 15 170 130 100 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 m M 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. F. Feng et al. Characterization of cathelicidins from C. coturnix FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 1579 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 (l M) Cc-CATH2 (0 m M 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 Characterization of cathelicidins from C. coturnix F. Feng et al. 1580 FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS SMARTÔ cDNA library construction kit (Clontech, Palo Alto, CA, USA). First-strand cDNA was synthesized by SMARTÔ IV oligonucleotide primer 5¢-AAGCAGTGG- TATCAACGCAGAGTGGCCATTACGGCCGGG-3¢ and CDS III ⁄ 3¢ PCR primer 5¢-ATTCTAGAGGCCGAGGC GGCCGACATGT (30)N –1 N-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 ⁄ H 2 O ⁄ 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 F. Feng et al. Characterization of cathelicidins from C. coturnix FEBS Journal 278 (2011) 1573–1584 ª 2011 The Authors Journal compilation ª 2011 FEBS 1581 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 · 10 5 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 A 595 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% CO 2 atmosphere at 37 °C. Cells (2 · 10 4 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]. IC 50 was defined as the concentration of Cc-CATHs at which A 490 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 A 540 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. 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Gene cloning, expression and characterization of avian cathelicidin orthologs, Cc-CATHs, from Coturnix coturnix Feifei Feng 1,2, *, Chen Chen 3, *,. 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,. 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