cathelicidin orthologs, Cc-CATHs, from Coturnix coturnix Feifei Feng1,2,*, Chen Chen3,*, Wenjuan Zhu2, Weiyu He1, Huijuan Guang2, Zheng Li2, Duo Wang1, Jingze Liu1, Ming Chen5, Yipeng Wa
Trang 1cathelicidin orthologs, Cc-CATHs, from Coturnix coturnix Feifei Feng1,2,*, Chen Chen3,*, Wenjuan Zhu2, Weiyu He1, Huijuan Guang2, Zheng Li2, Duo Wang1, Jingze Liu1, Ming Chen5, Yipeng Wang4and 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: 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 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 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.
Trang 2been 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, cathcath-elicidin 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 cathelicicathelici-dins 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
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
Trang 3(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.
Trang 4Cc-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 50C,
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 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.
Trang 5A 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
62
Pc-CATH1 62
Pc-CATH2 62
Pc-CATH3 62
Cc-CATH1 62
Cc-CATH2 62
Cc-CATH3 62
Fowlicidin1 62
Fowlicidin2 62
Fowlicidin3 72
Ea CATH1 72
Ec CATH1 72
Ec CATH2 72
Ec CATH3 73
Hs LL37 72
Ss PR39 72
Bt CATHL1 73
Oa SMAP29 72
Ch BAC5 73
Cp CAP11 70
Mm CRAMP 72
Oc CAP18 72
Clf K9CATH 68
Bf cath MLSCWVLVLALLGGACALPAP LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLGS MLSCWVLVLALLGGVCALPAP LSYPQALTQAVDSYNQRPELQ.NAFRLLSADPEPGPG.VDLST MLSCWVLVLALLGGACALPAP LDYNQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS MLSCWVLVLALLGGVCALPAP LSYPQALIQAVDTYNQRPEAQ.NAFRLLSADPEPGPG.IDLNT MLSCWVLVLALLGGACALPAP LDYNQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS MLSCWVLLLALLGGACALPAP LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS MLSCWVLLLALLGGVCALPAP LSYPQALIQAVDSYNQRPEVQ.NAFRLLSADPEPGPG.VDLST MLSCWVLLLALLGGACALPAP LGYSQALAQAVDSYNQRPEVQ.NAFRLLSADPEPGPN.VQLSS METQRNTRCLGRWSPLLLLLGLVIPPATT.QALSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKGD.KDSDT METQRDSCSLGRWSLLLLLLGLVIPLATT.QTLSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKED.EDPDT METQRNTRCLGRWSPLLLLLGLVIPPATT.QALSYKEAVLRAVDGLNQRSSDE.NLYRLLELDPLPKGD.KDSDT MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDA.NLYRLLDLDPRPTMD.GDPDT METQRASLCLGRWSLWLLLLGLVVPSAST.QALSYREAVLRAVDRLNEQSSEA.NLYRLLELDQPPKAD.EDPGT METPRASLSLGRWSLWLLLLGLALPSASA.QALSYREAVLRAVDQLNEQSSEP.NIYRLLELDQPP.QDDEDPDS 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 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 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 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 122 Pc-CATH1 122 Pc-CATH2 122 Pc-CATH3 122 Cc-CATH1 122 Cc-CATH2 122 Cc-CATH3 122 Fowlicidin1 122 Fowlicidin2 122 Fowlicidin3 130 Ea CATH1 130 Ec CATH1 130 Ec CATH2 130 Ec CATH3 133 Hs LL37 130 Ss PR39 143 Bt CATHL1 131 Oa SMAP29 130 Ch BAC5 131 Cp CAP11 139 Mm CRAMP 134 Oc CAP18 134 Clf K9CATH 143 Bf cath LHNLNFTIIETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRATFDVTCVESVADPV
LRTLNFTIMETECVPRAQTPIDDCDFKENGVIRDCSGPVTILQDTPEINLRCRDASSDPV
LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRATFDVTCVDSMADPV
LHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV
LRELNFTIMETECVPSARTRIDDCDFKENGAIKDCSGPVTILQDAPEISLNCRDASSDPV
LHNLNFTIMETRCQARSGAQLESCDFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV
LRALNFTIMETECTPSARLPVDDCDFKENGVIRDCSGPVSVLQDTPEINLRCRDASSDPV
LHNLNFTIMETRCQARSGAQLDSCEFKEDGLVKDCAAPVVLQGGRAVLDVTCVDSMADPV
PKPVSFTVKETVCPRTTQQPLEQCDFKENGLVKQCVGTVILDPVKASVDIGCDEPQRV
PKPVSFMVKETVCPRIMKQTPEQCDFKENGLVKQCVGTVILGPVKDHFDVSCGEPQRV
PKPVSFTVKETVCPRTTQQPLEECDFKENGLVKQCVGTVVLDPAKDYFDISCDKPQPI
PKPVSFMVKETVCPRIMKQTPEQCDFKENGLVKQCVGTVILDPVKDYFDASCDEPQRV
PKPVSFTVKETVCPRPTQRPPELCDFKENGRVKQCVGTVTLNPSNDPLDISCNEIQSV
PKRVSFRVKETVCSRTTQQPPEQCDFKENGLLKRCEGTVTLDQVRGNFDITCNNHQSIRITKQPWAPPQAA
PKPVSFRVKETVCPRTSQQPAEQCDFKENGLLKECVGTVTLDQVGNNFDITCAEPQSV
RKPVSFTVKETVCPRTTQQPPEECDFKENGLVKQCVGTVTLDPSNDQFDINCNELQSV
PKPVSFTIKETVCTKMLQRPLEQCDFKENGLVQRCTGTVTLDSAFNVSSLSCLGGRRF
PKSVRFRVKETVCGKAERQLPEQCAFKEQGVVKQCMGAVTLNPAADSFDISCNEPGAQPFRFKKISRLA
PKPVSFTVKETVCPKTTQQPLEQCGFKDNGLVKQCEGTVILDEDTGYFDLNCDSILQVKKID
NQELNFTMKETVCLVAEERSLEECDFQEDGVVMGCTGYYFFGESPPVVVLTCKPVGEEGEQKQEEGNEEEKEVEE
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
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
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
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
148 Pc-CATH1
154 Pc-CATH2
151 Pc-CATH3
148 Cc-CATH1
154 Cc-CATH2
151 Cc-CATH3
148 Fowlicidin1
154 Fowlicidin2
151 Fowlicidin3
155
Ea CATH1
156
Ec CATH1
157
Ec CATH2
170
Ec CATH3
170
Hs LL37
172
Ss PR39
155
Bt CATHL1
160
Oa SMAP29
176
Ch BAC5
177
Cp CAP11
172
Mm CRAMP
171
Oc CAP18
172 Clf K9CATH
191
Bf 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.
Trang 6higher 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, inclucathelici-ding the signal
B
Fig 3 (Continued).
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.
Trang 7peptide, 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 fowlicifowlici-din-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 Creator
kDa
21.7 kDa
kDa 1 2 3 4 0 5 6 7 8
1 0 kDa 100 80 50
30
20 12
170
C
130 100 70 55 40 35 25
15
170 100 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 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.
Trang 8Table 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
Gram-positive
Staphylococcus aureus
ATCC2592
Staphylococcus haemolyticus
(IS 2401, DRa)
Propionibacterium acnes
ATCC 11827
Gram-negative
Acinetobacter baumannii
(IS 2178, DRb)
Pseudomonas aeruginosa
ATCC 27853
Fungi
Trang 9SMART cDNA library construction kit (Clontech, Palo
Alto, CA, USA) First-strand cDNA was synthesized by
SMART IV oligonucleotide primer
5¢-AAGCAGTGG-TATCAACGCAGAGTGGCCATTACGGCCGGG-3¢ and
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
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: 94C for 1 min; 20
cycles of 94C for 20 s, 60 C for 30 s, 72 C for 60 s;
fol-lowed by a final extension at 72C for 5 min The second
part comprised: 94C for 3 min; 25 cycles of 94 C for
20 s, 58C for 30 s, 72 C for 60 s; followed by a final
extension at 72C 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 94C, 30 s at 64 C and 10 s at
72C followed by a final extension at 72 C for 10 min
GACCCGCTCGT-3¢ and the second reverse primer was 5¢-CCCAAGCTTCTAGCCAAAGCGTG-3¢ PCR comprised:
30 cycles of 30 s at 94C, 30 s at 64 C and 10 s at 72 C, followed by a final extension at 72C 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 correspondincreas-ing to the major peak were collected and lyophilized Subsequently, the anti-bacterial activity of expressed Cc-CATH2 with respect to
S aureusATCC2592 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
Trang 10analyzed 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 37C 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 37C for
18 h for bacteria and 48 h for fungus, and A595was
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 37C Cells
(2· 104per 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 37C 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 A490was 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 37C for 30 min
and centrifuged at 652 g for 5 min and A540of 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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