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

Novel cathelicidin-derived antimicrobial peptides from Equus asinus Zekuan Lu 1 *, Yipeng Wang 2,3 *, Lei Zhai 1 , Qiaolin Che 3 , Hui Wang 1 , Shuyuan Du 1 , Duo Wang 1 , Feifei Feng 1,2 , Jingze Liu 1 , Ren Lai 3 and Haining Yu 1,2 1 College of Life Sciences, Hebei Normal University, Shijiazhuang, China 2 School of Life Science and Biotechnology, Dalian University of Technology, China 3 Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China Introduction Cathelicidins are a family of structurally diverse anti- microbial peptides found in virtually all species of mammals that play a critical role in the innate immune system [1,2]. They are characterized by a N-terminal signal peptide (30 residues) and a highly conserved cathelin domain (99–114 residues long) followed by a C-terminal mature peptide (12–100 residues) that is characterized by a remarkable structural variety [3]. Cathelicidins are most abundantly present in circulat- ing neutrophils and myeloid bone marrow cells [4], Keywords cathelicidin; Equus asinus; function; gene cloning; peptide identification Correspondence R. Lai, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China Fax ⁄ Tel: +86 871 5196202 E-mail: rlai@mail.kiz.ac.cn H. Yu, College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050016, China Fax ⁄ Tel: +86 311 86268842 E-mail: yuhaining@dlut.edu.cn *These authors contributed equally to this work (Received 11 January 2010, revised 10 March 2010, accepted 15 March 2010) doi:10.1111/j.1742-4658.2010.07648.x In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory con- centration values against Gram-positive bacteria were mostly in the range of 0.3–2.4 lgÆmL )1 . EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 lgÆmL )1 . CD spectra showed that EA-CATH1 mainly adopts an a-helical conformation in a 50% trifluoroethanol ⁄ water solu- tion, but a random coil in aqueous solution. Scanning electron micro- scope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl 2 , EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread. Abbreviations cfu, colony-forming units; MH, Mueller–Hinton broth; MIC, minimal inhibitory concentration; SEM, scanning electron microscope. FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2329 and are also found in mucosal epithelial cells and skin keratinocytes [5]. To date, a number of cathelicidins have been identi- fied from mammals, such as humans, monkeys, mice, rats, rabbits, guinea pigs, pigs, cattle, sheep, goats and horses [6–8]. According to secondary structures, these cathelicidins are further divided into three groups [6]. Group one possesses an amphipathic a-helical struc- ture (human, mouse and bovine BMAP-34 peptides). Group two, including porcine PR-39 and bovine bacte- necins, is characterized by a high content of one or two amino acids, often proline and arginine. The third group mainly adopts a b-sheet structure, such as in protegrins. Apart from the primary antimicrobial activities, certain cathelicidins also participate in wound repair, the induction of angiogenesis and cytolysis, chemo- taxis for neutrophils, monocytes, mast cells and T cells [6,9]. Human cathelicidin LL-37 was reported to have antitumour and anti-HIV activities [10]. Con- cordant with these important roles in host defence and disease resistance, the aberrant expression of cathelicidins is often associated with various disease processes [11]. Therefore, future studies on the biological activities and clinical purposes of cathelici- dins will undoubtedly facilitate the treatment of infectious diseases, in addition to offering more novel therapeutic agents to stop the continued emergence of antibiotic resistance. The exact antimicrobial mechanism of cathelicidin is not clearly compre- hended. However, it is generally believed that its physical interactions with the negatively charged microbial membrane (phospholipids) resulting in membrane disruption is mainly responsible for its antimicrobial activity. Here we report the molecular cloning, identification and functional analysis of the cathelicidin from donkey (Equus asinus). Two cathelicidin-encoding cDNAs, one having a complete coding region (EA-CATH1) and the other only covering the mature peptide region (EA-CATH2), were cloned from the constructed lung cDNA library of donkey. The deduced mature antimi- crobial peptide EA-CATH1 was synthesized, and an array of functional activities, including antimicrobial, haemolytic and erythrocyte haemagglutination, were examined. Furthermore, the bacterial killing kinetics and factors related to antimicrobial activity (serum sta- bility, pH value) were also investigated. To better understand the mechanism of bactericidal action, the solution structure of EA-CATH1 was determined using CD spectroscopy and the effects on bacterial cell morphology were tested using scanning electron microscopy (SEM). Results and Discussion Identification and characterization of donkey cathelicidins We simultaneously constructed cDNA libraries of jugular lymph, penis, testis, lung, liver, spleen and bone marrow from donkey. Among them, the lung cDNA library was of the best quality, from which some posi- tive clones containing an insert of 555 bp were identi- fied and isolated. The nucleotide sequence of cDNA (from the start codon) (GenBank accession FJ803910) and deduced amino acid sequence of EA-CATH1 pre- cursor are shown in Fig. 1. Meanwhile, a clone with an insert of 450 bp was also sequenced, but lacked a signal peptide and partial cathelin domain. Using BLAST it was found that the cDNA coding region of EA-CATH1 displayed maximal 93% identity to the myeloid cathelicidin 2 (ECATH-2) of Equus caballus (GenBank accession NM001081869). The EA-CATH1 precursor was composed of 155 amino acid residues, including a predicted signal peptide, a conserved cathelin domain and the mature antimicrobial peptide EA-CATH1 (Fig. 1). Similar to other cathelicidins identified to date, prepro-EA-CATH1 also contained four cysteine residues in the conserved region [12] (Fig. 2). The processing of cathelicidin to generate mature antimicrobial peptides has been studied both in vitro and in vivo. Upon stimulation, the prepropeptide is processed to release the cathelin domain and the mature peptide. Elastase is generally considered to be responsible for such processing in fish, bird and mam- mals. Valine and alanine represent the most common elastase-sensitive residues [13]. Here, the valine (130) of prepro-EA-CATH1 is assumed to be the processing site by donkey elastase-like protease. Thereby, two mature peptides were predicted: EA-CATH1 (25 amino acids), KRRGSVTTRYQFLMIHLLRPKKLFA, and EA-CATH2 (26 amino acids), KGRGSETTRYQFV- PVHFFPWNKLSDF. Using BLAST they were found to be quite divergent from other mammalian cathelici- dins, even those characterized from horse. Analysis using the protparam tool (http://au.expasy.org/tools/ protparam.html) showed that the theoretical pI ⁄ Mw for EA-CATH1 and EA-CATH2 are 12.02 ⁄ 3060.75 and 9.70 ⁄ 3144.54, respectively. EA-CATH1 is a basic peptide smaller than most of the other cathelicidins identified to date. It comprises seven basic residues (four arginine and three lysine) with a net charge of 7. Thus, EA-CATH1 would be readily attracted by and adhere to the negatively charged bacterial surface to exert its potent antimicrobial activity. Characterization of cathelicidin from Equus asinus Z. Lu et al. 2330 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS Phylogenetic relationship between EA-CATH1 and other cathelicidins Multisequence alignment was performed on the basis of the full sequence of all cathelicidins. A condensed multifurcating tree was constructed emphasizing the reliable portion of pattern branches without consider- ing the exact distance between each peptide. Thus, the branch lengths of the condensed tree are not proportional to the number of amino acid mutations. The built phylogenetic tree revealed that vertebrate cathelicidins are split into two major clusters, and the sister group is represented by CATH37 from hagfish in a separated clade, which was potentially considered as an ancient member in the cathelicidin evolution. The second cluster is divided into two major groups: one represented by Atlantic cod, rain- bow trout; the other represented by snake cathelici- dins, avian fowlicidins and the most divergent mammalian cathelicidin families. Supported by a bootstrap value of 79%, EA-CATH1 was clustered with horse eCATH-1 and -3 (Fig. 3). Antimicrobial activity and bacteria killing kinetics Putatively mature EA-CATH1 was commercially syn- thesized and purified to > 95% purity. As listed in Table 1, EA-CATH1 showed broad-spectrum antimi- crobial activities against the tested micro-organisms, especially clinically isolated drug-resistant strains. In all antimicrobial assays, LL-37 characterized from human was used as the positive control. It is one of the most extensively studied cathelicidins so far. Com- pared with minimal inhibitory concentrations (MICs) of LL-37, EA-CATH1 showed much stronger antibac- terial potency. Among all 32 strains, Gram-positive bacterial strains were much more sensitive to EA-CATH1 than Gram-negative strains and fungus, with most MIC values in the range of 0.6–4.7 lgÆmL )1 (Table 1). EA-CATH1 even had a potent killing effect on the strains that were totally resistant to the conven- tional antibiotic drugs, e.g. Enterococcus faecium (IS091299) (MIC 9.4 lgÆmL )1 ). EA-CATH1 showed the strongest antimicrobial activity against Staphylo- coccus aureus ATCC2592 and S. haemolyticus 092401 with MICs as low as 0.6 lgÆmL )1 . For clinically iso- lated S. aureus and Nocardia asteroids, the MICs were both determined to be only 1.2 lgÆmL )1 . Interestingly, we also tested the antimicrobial activity of EA-CATH1 against Propionibacterium acnes, one kind of bacteria bothering a large population all over the world. EA-CATH1 also had a fairly small MIC of 4.7 lgÆmL )1 . However, half of the Gram-negative bac- teria tested seemed not to be very sensitive to EA-CATH1 and LL-37 performed even worse. The killing kinetics of EA-CATH1 were examined using a colony counting assay, with ampicillin as the positive control. As listed in Table 2, EA-CATH1 exerted antibacterial activity in a faster kinetics than ampicillin. It could rapidly kill S. aureus (ATCC2592), with the maximum killing occurring at less than 0.5 h (versus 2 h for ampicillin) at 10· MIC; 1 h (versus 3 h Fig. 1. The cDNA sequence encoding EA-CATH1 and the predicted prepropeptide sequence. The signal peptide predicted by SIGNALP 3.0 is shaded in grey. The putative mature peptide of EA-CATH1 is boxed. The stop codon is indicated by an asterisk. The 3¢- UTR is in lowercase letters. The potential polyadenylation signal (aaaaataaa) is underlined. Z. Lu et al. Characterization of cathelicidin from Equus asinus FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2331 for ampicillin) at 5· MIC and 2 h (versus 6 h for ampicillin) at 1· MIC. The antibacterial activity proved to be lethal for S. aureus ATCC2592. Staphylo- coccus aureus was not capable of resuming growth on agar plates after a 2 h treatment with concentrations above the corresponding MICs. In contrast, ampicillin could not clean the bacteria within 2 h. EA-CATH1 of 5· MIC killed micro-organisms almost five times faster than 1· MIC (Table 2). Secondary structures of EA-CATH1 and the effects on bacterial cell morphology The CD spectrum of EA-CATH1 in water showed a negative band at 200 nm, indicating a random coil conformation. In a membrane-mimetic solvent such as 50% trifluoroethanol ⁄ water, the presence of one posi- tive band (190 nm) and two negative dichroic bands at 208 and 222 nm are consistent with the a-helical con- formation (Fig. 4). The current result is in good agree- ment with the online prediction by GOR IV (http:// npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_ gor4.html), which showed a 36% a-helical peptide (Y9-L17) in the middle, a 56% random coil (K1-R8, R18-K22, A25) on both sides of the a-helix and two amino acid extended stands (L23, F24) close to the C-terminus. The a-helical structure of most active cathelicidin peptides is thought to be responsible for the formation of pores in the membranes of target organisms, thus disrupting metabolic activity [14]. This is also approved by LL-37 [15]. Its helical, oligo- meric conformation is required for potent antibacterial Fig. 2. Multiple sequence alignment of EA-CATH1 with representative cathelicidins; conserved residues are shaded. The four conserved cysteine residues in cathelin domain are framed. Each mature cathelicidin is underlined. Ea, Equus asinus (donkey); Ec, Equus caballus (horse) [12]; Clf, Canis lupus familiars (dog) [27]; Bt, Bos taurus (cattle) [28]; Oa, Ovis aries (sheep) [29]; Ch, Capra hircus (goat) [30]; Ss, Sus scrofa (pig) [31]; Hs, Homo sapiens (human) [7]; Oc, Oryctolagus cuniculus (rabbit) [32]; Mm, Mus musculus (mouse) [33]; Cp, Cavia porcellus (guinea pig) [34]; Gg, Gallus gallus (chicken) [8]; Me, Macropus eugenii (tammar wallaby) [35]; Bf, Bungarus fasciatus (snake) [22]. Characterization of cathelicidin from Equus asinus Z. Lu et al. 2332 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 98 99 64 97 98 92 100 100 100 95 73 70 94 81 78 92 83 56 98 56 90 66 100 70 83 79 98 69 63 100 61 78 98 100 100 100 98 Fig. 3. Phylogenetic analysis of representa- tive vertebrate cathelicidins. The phyloge- netic dendrogram was constructed using the neighbour-joining method based on the proportion difference of aligned amino acid sites of the full sequence of prepropeptide. Only bootstrap values > 50% (expressed as a percentage of 1000 bootstrap samples supporting the branch) are shown at branching points. The bar indicates the branch length. Z. Lu et al. Characterization of cathelicidin from Equus asinus FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2333 activity. The CD result supports the conception that EA-CATH1 probably kills bacteria through membrane disruption. A generally acknowledged antimicrobial mechanism of cathelicidin is its physical interactions with the neg- atively charged microbial membrane, followed by membrane lysis [16]. Such an interaction is often directly correlated with the extent of antibacterial activity and makes it hard to develop resistance [17]. In the present study, the effects of EA-CATH1 on the cellular morphology of S. aureus were observed by SEM. Control cells with no peptide treatment exhib- ited a normal shape and smooth surfaces (Fig. 5A). In contrast, treatment with EA-CATH1 for 30 min severely disrupted the cell wall and cell membrane of S. aureus (Fig. 5B–D). During treatment, the bacterial cells appeared to have a rough surface, with crimpled and bent morphologies (Fig. 5B–D), and were then finally lysed. Haemolysis, serum stability and the effect of pH on antimicrobial activity A big problem commonly associated with clinical applications of cathelicidins is their haemolysation of mammalian cells. However, the good thing is that the dose of cathelicidin resulting in haemolysis is often much higher than the MIC. The haemolytic capability Table 1. Antimicrobial activity of EA-CATH1. These concentrations represent the mean values of three independent experiments performed in duplicate. ND, no detectable activity in the inhibition zone assay at a dose of 2 mgÆmL )1 ; > 100, detectable antimicrobial activity in the inhibition zone assay, but did not totally inhibit cell growth in liquid medium at a dose up to 100 lgÆmL )1 ; IS, clinically isolated strain; DRa, drug resistance for ceftazidime, cefoperazone and aztreonam; DRb, drug resistance for compound sulfamethoxazole, erythromycin, ciproflox- acin and penicillin. Micro-organism MIC (lgÆmL )1 ) EA-CATH1 LL-37 Ampicillin Kanamycin Gram positive Staphylococcus aureus (IS) 1.2 18.8 4.7 75 Staphylococcus aureus ATCC2592 0.6 4.7 2.4 4.7 Staphylococcus haemolyticus (092401) (IS, DRa) 0.6 ND 0.3 ND Nocardia asteroids (IS) 1.2 18.8 1.2 75 Enterococcus faecium (IS091299) 9.4 18.8 ND ND Propionibacterium acnes ATCC11827 4.7 > 100 1.2 2.4 Gram negative Klebsiella oxytoca (IS) > 100 ND ND ND Aeromonas sobria (IS) 9.4 18.8 ND ND Acinetobacter baumannii 092178 (IS, DRb) 4.7 ND 37.5 2.4 Acinetobacter baumannii 092373 (IS) 9.4 ND ND ND Stenotrophomonas maltophilia (IS) 9.4 18.8 ND ND Pseudomonas aeruginosa ATCC27853 > 100 ND ND ND Pseudomonas aeruginosa 091411(IS) 18.8 > 100 ND ND Pseudomonas aeruginosa 091412 (IS) > 100 ND ND ND Pseudomonas aeruginosa 091413 (IS) > 100 ND > 100 75 Escherichia coli 091335 (IS) 75 ND ND ND Escherichia coli ATCC25922 ND ND 9.4 9.4 Escherichia coli 090223 (IS) ND ND 75 2.4 Serratia marcescens 091379 (IS) ND ND ND ND Klebsiella pneumoniae 091372 (IS) > 100 ND ND ND Klebsiella pneumoniae 091373 (IS) > 100 ND ND 2.4 Klebsiella pneumoniae 091400 (IS) ND ND ND ND Proteus vulgaris (IS) > 100 > 100 1.2 4.7 Proteus mirabilis (IS) > 100 ND 2.4 4.7 Salmonella typhi 091408 (IS) ND ND ND 18.8 Fungi Candida albicans 092251 (IS) 9.4 > 100 4.7 18.8 Candida albicans ATCC2002 9.4 9.4 0.6 1.2 Slime mould 4.7 18.8 2.4 75 Candida glabrata 090902 (IS) > 100 ND ND ND Candida tropicalis 092422 (IS) > 100 ND ND ND Cryptococcus neoformans (IS) ND ND ND ND Characterization of cathelicidin from Equus asinus Z. Lu et al. 2334 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS of EA-CATH1 was tested using freshly prepared human erythrocytes. The result indicated that EA-CATH1 (20 lgÆmL )1 ) had almost no haemolytic activity (1.8%) on human red blood cells in a dose much higher than the MIC. Thus, EA-CATH1 showed considerable selectivity for micro-organisms over mam- malian cells in vitro. The serum stability of EA-CATH1 was also exam- ined; the results are listed in Table 3. To our surprise, after incubating with 90% fresh human serum for up to 72 h, EA-CATH1 still retained strong antimicrobial activity against S. aureus, much longer than other Table 2. Bacterial killing kinetics of EA-CATH1. Time cfu (Staphylococcus aureus ATCC2592) 0 min 10 min 30 min 1 h 1.5 h 2 h 3 h 6 h EA-CATH1(·1 MIC a )63 44 21 9 1 0 0 0 EA-CATH1(·5 MIC) 57 22 3 0 0 0 0 0 EA-CATH1(·10 MIC) 54 5 0 0 0 0 0 0 Ampicillin (·1 MIC b )67 62 42 19 9 4 1 0 Ampicillin (·5 MIC) 61 46 23 6 3 1 0 0 Ampicillin (·10 MIC) 60 37 13 4 1 0 0 0 Water 68 87 113 177 264 432 1280 17 175 cfu (Acinetobacter baumannii 092178 IS) EA-CATH1(·1 MIC c )61 55 49 38 28 12 1 0 EA-CATH1(·5 MIC) 57 46 39 22 5 0 0 0 EA-CATH1(·10 MIC) 55 41 33 15 0 0 0 0 Water 62 81 148 234 355 591 1706 22 724 a EA-CATH1 MIC to S. aureus ATCC2592 0.6 lgÆmL )1 ; b ampicillin MIC to S. aureus ATCC2592 2.4 lgÆmL )1 ; c EA-CATH1 MIC to A. baumannii (092178 IS) 4.7 lgÆmL )1 . 100 EA-CATH1 40 60 80 Water TFE 50% –20 0 20 CD [mdeg] –80 –60 –40 190 200 210 220 230 240 250 Wavelen g th [nm] Fig. 4. CD analysis of EA-CATH1 in trifluoroethanol ⁄ water (50% v ⁄ v). 9051910 1.0 kV 7.4 mm x20.0k SE(M) 5/20/2009 09:40 905197 1.0 kV 7.6 mm x20.0k SE(M) 5/20/2009 10:34 905198 1.0 kV 7.6 mm x20.0k SE(M) 5/20/2009 10:13 905197 1.0 kV 7.6 mm x20.0k SE(M) 5/20/2009 10:31 1xMIC 10xMIC 10xMIC 2.00 µm 2.00 µm 2.00 µm 2.00 µm AB CD Fig. 5. SEM of Staphylococcus aureus trea- ted with EA-CATH1. (A) Control S. aureus; (B) S. aureus treated with EA-CATH1 at 1· MIC; (C, D) S. aureus treated with EA-CATH1 at 10· MIC. Z. Lu et al. Characterization of cathelicidin from Equus asinus FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2335 cathelicidins [18]. Such extraordinary stability in serum implies the potential of EA-CATH1 for systemic thera- peutic applications. More interestingly, during the first 3 h after adding EA-CATH1 to serum, the MIC (0.6 lgÆmL )1 ) against S. aureus was lower by half than in water. This might be due to the antibacterial activity of serum proteins, which lately has been given a lot of attention. Or, possibly, our peptide is highly serum protein bound, which could lead to the conformational change (a more helical structure) and would explain the lower MICs in the presence of the serum. The MICs of EA-CATH1 incubating with human serum for 3–12, 24–48 and 60–72 h were 1.2, 4.7 and 9.4 lgÆmL )1 , respectively. The effects of pH on the antimicrobial activity of EA-CATH1 were tested (Table 4). Clearly, in the pH range of 5.0–9.0, the acidic pH (5.0–7.0) benefited the antimicrobial effect against S. aureus (Gram positive), whereas Acinetobacter baumannii (Gram negative) and Candida albicans (fungus) were more sensitive to EA-CATH1 at the basic pH (7.0–9.0). Among the three strains, the MIC for C. albicans was influenced most, varying from 2.4 to 18.8 lgÆmL )1 under a corre- sponding pH from 9.0 to 5.0. At optimal pH values around 7.0, EA-CATH1 showed the strongest antimi- crobial activities with the lowest MICs. The explana- tion for such pH-dependent activity is the pH-induced structural changes in peptide conformation. The a-heli- cal structure is thought to be important for the antimi- crobial activity of cathelicidins [14], and its content is usually unchanged over the neutral pH range, but is drastically reduced at higher or lower pH values. Thereby, the pH-induced peptide unfolding may con- tribute to the reduced activity of EA-CATH1 at acidic or basic pH values. The assay against C. albicans might have involved certain inevitable error resulting in a slightly higher optimal pH (8.0). The other expla- nation is that EA-CATH1 might exert its antifungal activity through the formation of reactive oxygen spe- cies [19]. This process is irrelevant to peptide solution structure, thus in turn irrelevant to pH. Erythrocytes haemagglutination activity EA-CATH1 had no detected haemagglutination activ- ity on fresh rabbit erythrocytes in the assay. However, in the presence of CaCl 2 , it could exert an agglutina- tion activity with the minimum concentration of 50 lgÆmL )1 (16.3 lm). So far, significant peptide- induced haemagglutination has been observed for certain cathelicidins, such as LL-37 (‡ 25 lm) and indolicidin (‡ 100 lm) [14]. It has been proposed that the bacteria secreted or membrane-bound polyproteins can bind to, agglutinate and lyse local host erythro- cytes [20]. Thus, the cationic cathelicidins might poten- tiate an inhibition against the electrostatic interaction between the bacterial polyproteins and the haemag- glutinin binding domains on the erythrocyte surface [21]. It has been reported that antimicrobial peptides, including cathelicidin LL-37, were effective in disrupting Porphyromonas gingivalis-induced haema- gglutination among erythrocytes [22]. Therefore, the haemagglutination ability of EA-CATH1 in the pres- ence of CaCl 2 makes it a good drug candidate to potentially restrict bacterial colonization and spread by the perturbation of bacterial polyproteins. In summary, in the present work, EA-CATH1 was identified by molecular cloning as a member of Table 3. Stability of EA-CATH1 in human serum. MIC (lgÆmL )1 ) Time (h) 036122436486072 Clinically isolated Staphylococcus aureus 0.6 1.2 1.2 1.2 4.7 4.7 4.7 9.4 9.4 Table 4. Antimicrobial activity of EA-CATH1 in 150 mM NaCl ⁄ P i at different pH values (mean values of three independent experiments performed in duplicate). IS, clinically isolated strain; DRb, drug resistance for compound sulfamethoxazole, erythromycin, ciprofloxacin and penicillin. –, S. aureus (IS) did not grow. Micro-organism MIC (lgÆmL )1 ) Water pH 5 pH 6 pH 7 pH 8 pH 9 Staphylococcus aureus (IS) 1.2 – 0.6 0.6 1.2 1.2 Acinetobacter baumannii 092178(IS, DRb) 4.7 9.4 2.4 4.7 4.7 4.7 Candida albicans ATCC2002 9.4 18.8 9.4 4.7 2.4 2.4 Characterization of cathelicidin from Equus asinus Z. Lu et al. 2336 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS cathelicidin-derived antimicrobial peptides from don- key (E. asinus). The nucleotide and deduced amino acid sequences of prepro-EA-CATH1 were compar- atively conserved among mammalian cathelicidin families. The chemically synthesized EA-CATH1 has broad-spectrum potent antibacterial activity, but no haemolytic activity in high doses, implying a promis- ing therapeutic potential. In addition, the human serum stability and haemagglutination capacity of EA-CATH1 makes it an excellent candidate for the development of novel antimicrobial and antisepsis agents. The results of a pH-dependency assay coupled with killing kinetics may offer important data for clinical studies. Materials and methods Collection of tissues Tissue samples of an adult male donkey were collected from Beijing Hongfa Donkey Meat Processing Plant (Beijing, China), including lung, spleen, liver, jugular lymph, testis, penis and bone marrow. The collection proce- dure was according to either routine management of the farm animals or surplus from other approved research pro- tocols. Tissues were dissected and frozen immediately in liquid nitrogen until used. Molecular cloning of cathelicidin and phylogenetic tree construction Total RNA was extracted from each tissue collected using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR-based cDNA was synthesized using the CreatorÔ SMARTÔ cDNA library construction kit (Clontech, Palo Alto, CA, USA) as described by the manufacturer. The first-strand cDNA was synthesized using PowerScript reverse transcriptase with the SMART TM IV oligonucleotide primer 5¢-AAGCAGTGGT ATCAACGCAGAGTGGCCATTACGGCCGGG-3¢ and the CDS III ⁄ 3¢ PCR primer 5¢-ATTCTAGAGGCCGA GGCGGCCGACA TGT(30)N -1 N-3¢ (N = A, G, C or T; N -1 = A, G or C). The second strand was amplified using Advantage DNA polymerase from Clontech with the 5¢ PCR primer 5¢-AAGCAGTGGTATCAACGCAGAGT-3¢ and the CDS III ⁄ 3¢ PCR primer. According to the conserved signal peptide domain of pre- viously characterized horse cathelicidin cDNA [23], two sense primers P1 (5¢-GGACCATGGAGACCCAGAGG-3¢) and P2 (5¢-ATGGAGACCCAGAGGGACAGTT-3¢) were designed from 5¢-UTR and a highly conserved domain- encoding part of the signal peptide of horse cathelicidin cDNAs and coupled with CDS III ⁄ 3¢ PCR primer. The half nested PCR conditions involved two sections. First section: 94 °C for 1 min; 25 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s; followed by a final extension at 72 °C for 10 min. Second section: 94 °C for 5 min; 30 cycles of 94 °C for 20 s, 58 °C for 20 s, 72 °C for 45 s; followed by a final extension at 72 °C for 10 min. The PCR product was purified by gel electrophoresis, cloned into pGEM-T vector (Pro- mega, Madison, WI, USA). DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377 (Perkin Elmer Corp., Norwalk, CT, USA). The phylogenetic tree was constructed with the neigh- bour-joining method using clustalw (version 1.8). Multi- cathelicidin sequences aligned were obtained from the protein database at the National Center for Biotechnology Information. CD spectroscopy The peptide used for the bioactivity test and CD spectros- copy was synthesized by the peptide synthesizer GL Biochem (Shanghai, China), and purified to > 95% purity. To investigate the secondary structure of EA-CATH1, CD spectroscopy was performed using a Jasco J-715 spectro- photometer. Samples with a constant peptide concentration of 0.5 mgÆmL )1 were prepared in two different solvents, water and 50% (v ⁄ v) trifluoroethanol ⁄ water, and added in a quartz optical cell with a path length of 0.5 mm at 25 °C. The spectra were averaged over three consecutive scans, followed by subtraction of the CD signal of the solvent. Antimicrobial assay and bacteria killing kinetics In total, 31 standard (purchased commercially) and clini- cally isolated bacterial and fungal strains (obtained from a local hospital) were used for the antimicrobial assays (Table 1). The assay was conducted as described previously [24]. The MIC was measured using the standard micro- dilution broth method in a 96-well microtitre plate. Serial dilutions (50 lL) of the peptides in Mueller–Hinton broth (MH) were prepared in 96-well microtitre plates and mixed with 50 lL bacteria inoculums in MH [1 · 10 6 colony- forming units (cfu)ÆmL )1 ]. The human cathelicidin LL-37 and the antibiotics ampicillin and kanamycin were used as positive controls. The microtitre plate was incubated at 37 °C for 18 h for bacteria and 48 h for fungal strains and absorbance was measured at 595 nm using a microtitre plate spectrophotometer. MIC was defined as the lowest concentration of peptide that completely inhibits growth of the microbe determined by visual inspection or spectro- photometrically the growth percentage was less than 5% compared with that of the negative control. The bactericidal effects of EA-CATH1 against S. aureus ATCC2592 (1 · 10 6 cfuÆmL )1 ) and A. baumannii (1 · 10 6 cfuÆmL )1 ) were tested at 1, 5 and 10· corresponding Z. Lu et al. Characterization of cathelicidin from Equus asinus FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS 2337 MICs, with ampicillin as the positive control. Fresh colo- nies of the bacteria were cultured overnight to log phase, measured absorbance at 600 nm (A 600 )is 8 · 10 8 cfuÆmL )1 and then diluted with fresh MH to 1 · 10 6 cfuÆmL )1 . EA-CATH1 was added to the bacterial suspen- sion, achieving the final sample concentration to 1, 5 and 10 · corresponding MICs. The mixture was incubated at 37 °C. Colony counting was performed at 0 min, 10 min, 30 min, 1 h, 1.5 h, 2 h, 3 h and 6 h [24]. At each time point, 1 lL mixture was diluted with MH to 1 mL, then 50 lL diluted bacterial suspension was plated out at 37 ° C for 12 h before colony counting. SEM A log phase culture (1 · 10 6 cfuÆmL )1 )ofS. aureus (ATCC2592) was incubated with EA-CATH1 (1 · , 10 · MIC) at 37 °C for 30 min. Aliquots of the cultures were fixed with 6% glutaraldehyde solution for 4 h. The bacteria were then centrifuged (300 g for 10 min) and washed with 0.1 m phosphate-buffered saline (NaCl ⁄ P i ), pH 7.2. The pellets were then fixed in 1% osmium tetroxide in 0.1 m NaCl ⁄ P i , pH 7.2 for 1 h. The cells were rinsed with the same buffer and dehydrated in a graded series of etha- nol and then frozen in liquid nitrogen-cooled tert-butyl alcohol and vacuum dried overnight. The samples were mounted on to aluminium stubs. After sputter coating with gold, they were analysed using a Hitachi S-4800 SEM. Haemolysis, serum stability and the effect of pH on antimicrobial activity Haemolysis assays were conducted as previously described [25]. The EA-CATH1 of 20 lgÆmL )1 was incubated with washed human erythrocytes at 37 °C for 30 min and centri- fuged at 1000 g for 5 min. Absorbance of the supernatant was measured at 540 nm. Triton X-100 (1% v ⁄ v) was used to determine the maximal haemolysis. The experiment was repeated three times. The serum stability of EA-CATH1 (2 mgÆmL )1 ) was examined by incubating with 90% freshly prepared human serum at 37 °C for 0, 3, 6, 12, 24, 36, 48, 60 and 72 h. The MIC was then recorded at each time interval. EA-CATH1 was dissolved in 150 mm NaCl ⁄ P i (sterilized by filter) at pH 4, 5, 6, 7, 8 and 9. The MICs of EA-CATH1 on Gram-positive bacterium S. aureus, Gram- negative bacterium A. baumannii and fungus C. albicans (ATCC2002) cultured in MH were then tested. Erythrocyte haemagglutination assay Fresh intact rabbit erythrocytes were prepared as previously described [26]. Assays were performed in 96 U-well micro- titre plates. The haemagglutinating activity of EA-CATH1 was determined by a two-fold serial dilution procedure using rabbit erythrocytes. The haemagglutination titre was defined as the reciprocal of the highest dilution exhibiting haemagglutination. To examine the divalent cation effect, 20 mm Tris ⁄ HCl (pH 7.5) with or without 50 mm CaCl 2 was used as the assay buffer. Acknowledgement We thank the editor and four anonymous reviewers for their helpful comments on the manuscript. This work was supported by grants from the Chinese National Natural Science Foundation (30900240). References 1 Zanetti M (2005) The role of cathelicidins in the innate host defenses of mammals. Curr Issues Mol Biol 7, 179– 196. 2 Ramanathan B, Davis EG, Ross CR & Blecha F (2002) Cathelicidins: microbiocidal activity, mechanisms of action, and roles in innate immunity. Microbes Infect 4, 361–372. 3 Zanetti M, Gennaro R, Scocchi M & Skerlavaj B (2000) Structure and biology of cathelicidins. Adv Exp Med Biol 479, 203–218. 4 Gennaro R & Zanetti M (2000) Structural features and biological activities of the cathelicidin-derived antimi- crobial peptides. Biopolymers 55, 31–49. 5 Zanetti M & Leukoc J (2004) Cathelicidins, multifunc- tional peptides of the innate immunity. Biology 75, 39–48. 6 Zaiou M & Gallo RL (2002) Cathelicidins, essential gene-encoded mammalian antibiotics. J Mol Med 80, 549–561. 7 Turner J, Cho Y, Dinh NN, Waring AJ & Lehrer RI (1998) Activities of LL-37, a cathelin-associated antimi- crobial peptide of human neutrophils. Antimicrob Agents Chemother 42, 2206–2214. 8 Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) Identification and functional characterization of three chicken cathelicidins with potent antimicrobial activity. J Biol Chem 281, 2858–2867. 9 Carretero M, Escamez MJ, Garcıa M, Duarte B, Holguın A, Retamosa L, Jorcano JL, Rı ´ oMD& Larcher F (2008) In vitro and in vivo wound healing promoting activities of human cathelicidin LL-37. J Invest Dermatol 128, 223–236. 10 Hancock RE & Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24, 1551–1557. 11 Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, Gallo RL & Leung DY (2002) Endogenous antimicrobial peptides and skin Characterization of cathelicidin from Equus asinus Z. Lu et al. 2338 FEBS Journal 277 (2010) 2329–2339 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... Peptidomics and genomics analysis of novel antimicrobial peptides from the frog, Rana nigrovittata Genomics 95, 66–71 Characterization of cathelicidin from Equus asinus 25 Bignami GS (1993) A rapid and sensitive hemolysis neutralization assay for palytoxin Toxicon 31, 817– 820 26 Kawagishi H, Nomura A, Mizuno T, Kimura A & Chiba S (1990) Isolation and characterization of a lectin from Grifola frondosa fruiting... Chem 273, 3718–3724 Zasloff M (2002) Antimicrobial peptides of multicellular organisms Nature 415, 389–395 Zanetti M, Gennaro R, Skerlavaj B, Tomasinsig L & Circo R (2002) Cathelicidin peptides as candidates for a novel class of antimicrobials Curr Pharm Des 8, 779– 793 Wang Y, Hong J, Liu X, Yang H, Liu R, Wu J, Wang A, Lin D & Lai R (2008) Snake cathelicidin from Bungarus fasciatus is a potent peptide... Cathelicidins – a family of multifunctional antimicrobial peptides Cell Mol Life Sci 60, 711–720 Shinnar AE, Butler KL & Park HJ (2003) Cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance Bioorg Chem 31, 425–436 Oren Z, Lerman JC, Gudmundsson GH, Agerberth B & Shai Y (1999) Structure and organization of the human antimicrobial peptide LL-37 in phospholipid... RI (1999) Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes Infect Immun 67, 4106–4111 31 Agerberth B, Lee JY, Bergman T, Carlquist M, Boman HG, Mutt V & Jornvall H (1999) Amino acid sequence ¨ of PR-39 Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides Eur J Biochem 202, 849–854 32 Larrick JW, Hirata... biochemical activity of a novel pro-myeloid antimicrobial peptide Dev Comp Immunol 31, 1278–1296 28 Del Sal G, Storici P, Schneider C, Romeo D & Zanetti M (1992) cDNA cloning of the neutrophil bactericidal peptide indolicidin Biochem Biophys Res Commun 187, 467–72 29 Mahoney MM, Lee AY, Brezinski-Caliguri DJ & Huttner KM (1995) Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide... synthetic histidine-rich peptides on haemagglutination by Bacteroides gingivalis 381 Arch Oral Biol 35, 775– 777 Dixon DR, Jeffrey NR, Dubey VS & Leung KP (2009) Antimicrobial peptide inhibition of Porphyromonas gingivalis 381-induced hemagglutination is improved with a synthetic decapeptide Peptides 30, 2161–2167 Scocchi M, Bontempo D, Boscolo S, Tomasinsig L, Giulotto E & Zanetti M (1999) Novel cathelicidins... JW, Hirata M, Zheng H, Zhong J, Bolin D, Cavaillon JM, Warren HS & Wright SC (1994) A novel granulocyte-derived peptide with lipopolysaccharideneutralizing activity J Immunol 152, 231–240 33 Gallo RL, Kim KJ, Bernfield M, Kozak CA, Zanetti M, Merluzzi L & Gennaro R (1997) Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse J Biol Chem 272, 13088–13093 . Novel cathelicidin-derived antimicrobial peptides from Equus asinus Zekuan Lu 1 *, Yipeng Wang 2,3 *, Lei. EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested