动 物 学 研 究 2009，Apr. 30(2): 171−177 CN 53-1040/Q ISSN 0254-5853 Zoological Research DOI：10.3724/SP.J.1141.2009.02171 Effects of the Antimicrobial Peptide OH-CATH on Escherichia coli CHEN Xin-xin1,2, YU Guo-yu1,3, ZHAN Yan2, ZHANG Yun 1, SHEN Ji-hong 4,*, LEE Wen-hui1, * (1. Key Laboratory of Animal Models and Human Diseases Mechanisms, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming Yunnan 650223, China; 2. Cell Biology and Genetics Section of Kunming Medical College, Kunming Yunnan 650031, China; 3. Biochemistry Section of Kunming Medical College, Kunming Yunnan 650031, China；4. Department of Urology, the First Affiliated Hospital of Kunming Medical College, Kunming Yunnan 650032, China) Abstract: OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a high concentration. OH-CATH might serve as model molecules for the development of antimicrobial drugs. Understanding the action mechanism of OH-CATH and the reason for its selectivity against microbes is very important for this purpose. The bactericidal effect of the king cobra antimicrobial peptide OH-CATH on Gram-negative Escherichia coli (ATCC 25922) is observed by scanning electron microscopy (SEM) and transmitted electron microscopy (TEM). The SEM and TEM results suggested that the bactericidal mechanism of OH-CATH against Escherichia coli happened in three steps. Firstly, OH-CATH attached to the negatively charged bacterial wall by positively charged amino acid residues. In the second step, the accumulated OH-CATH aggregated and damaged the bacteria membrane in a pore-forming manner. In the last step, with the damage of cell permeability, the contents of the cells were released and eventually cells died. Key words: Antimicrobial peptide; OH-CATH; Escherichia coli; Electron microscopy 抗菌肽 OH-CATH 对大肠杆菌 Escherichia coli 的作用 陈欣欣1,2，余果宇3，张 彦2，张 云1，申吉泓4,*，李文辉1,* (1. 中国科学院动物模型与人类疾病机理重点实验室，云南 昆明 650223；2. 昆明医学院 细胞生物学及遗传学教研室，云南 昆明 650031；3. 昆明医学院 生物化学教研室，云南 昆明 650031；4. 昆明医学院第一附属医院 泌尿外科，云南 昆明 650032) 摘要：OH-CATH是眼镜王蛇中新发现的cathelicidin家族抗菌肽。它在1% NaCl存在的条件下对多种细菌都有 较强的抗菌活性，同时，在高浓度下对人红细胞无溶血活性。OH-CATH是开发新型抗菌药物的优良模板。阐明 OH-CATH的作用机理及其对微生物的选择性，对研发以OH-CATH为先导结构的药物研发有十分重要的意义。本 文利用扫描电镜以及透射电镜对OH-CATH与革兰氏阴性菌－大肠杆菌ATCC 25922相互作用的效应研究。结果揭 示：OH-CATH对大肠杆菌的作用涉及到3个步骤。首先，OH-CATH借助其带正电的氨基酸残基附着到细菌带负电 荷的细胞壁；然后，附着的OH-CATH在达到一定浓度后发生聚集，以孔道形成的方式破坏细菌的膜结构；最终， 由于细菌膜的损坏，膜的渗透性被破坏，胞内内含物释放造成细菌死亡。 关键词：抗菌肽；OH-CATH；大肠杆菌；电镜 中图分类号：Q514.3; Q939.92 文献识码：A 文章编号：0254-5853-(2009)02-0171-07 It has been widely recognized that antimicrobial (Broekaert et al, 1995), invertebrates (Vizioli & Salzet, peptides play an important role in protecting organisms 2002), vertebrates (Hancock & Lehrer, 1998; Li et al, from invading of microorganisms (Boman, 1991, 1995; 2001), and human (Harder et al, 1997; Li et al, 1995; Li Zasloff, 1992; Nicolas & Mor, 1995). Antimicrobial et al, 2001; Zhao et al, 2008a,b). Because of the growing peptides have successfully been isolated from plants problem of multi-drug resistant pathogenic organisms, Received date: 2008-11-26; Accepted date: 2009-02-13 收稿日期：2008-11-26；接受日期：2009-02-13 基金项目：国家自然科学基金（30670412）；中国科学院重要方向项目（KSCX2-YW-R-088）；云南省自然科学基金资助项目 （2005PY01-23；2006PT08-1；2006C0047Q） *通讯作者(Corresponding authors), 申吉泓，Tel: 0871-5324888-2402，E-mail: [email protected]；李文辉，Tel: 0871-5194279, E-mail: [email protected] 第一作者简介：陈欣欣（1972-），女，硕士研究生。研究方向：生物化学与分子生物学 172 Zoological Research Vol. 30 there is increased interest in the pharmacological In the present work, the bactericidal effect of the application of antimicrobial peptides to treat infection. king cobra antimicrobial peptide OH-CATH on Currently, large efforts are being made to increase the Gram-negative Escherichia coli (ATCC 25922) is potency and specificity of these peptides so that they observed by scanning electron microscopy (SEM) and become toxic to microbes and not to humans. In order to transmitted electron microscopy (TEM). The bactericidal achieve this goal in an efficient manner, it is important to mechanism of OH-CATH against E. coli was reported elucidate the mechanism of action of these peptides and based on the obtained SEM and TEM images. the reason for their selectivity against microbes (da Silva 1 Materials and Methods & Teschke, 2003). Cathelicidins are cationic host defense peptides that 1.1 Materials play an important role in innate immune system. Escherichia coli ATCC 25922 was obtained from Members of the cathelicidin family all include The First Affiliated Hospital of Kunming Medical N-terminal signal peptides and proregions highly College, China. Yeast extract and Tryptone for bacterial homologous to cathelin, a cathepsin L inhibitor, then culture were products of Oxoid Ltd. Company (England). followed by highly divergent C-terminal antimicrobial All other reagents used were analytic grade from domains (Bals & Wilson, 2003; Lehrer & Ganz, 2002). commercial sources. Up to now，most of the identified cathelicidins were from 1.2 Peptide synthesis mammalian species (Zanetti et al, 1990, 1993, 1995). King cobra antimicrobial peptide OH-CATH with Five cathelicidins termed fowlicidin-1, 2, 3, B1 and amino acid sequence of KRFKKFFKKLKNSVKKRAK- myeloid antimicrobial peptide 27 were characterized KFFKKPRVIGVSIPF was synthesized by solid-phase from chicken (Xiao et al, 2006; Goitsuka et al, 2007). synthesis on an Applied Biosystems Model 433A peptide Cathelicidins were also found in fish species of Atlantic synthesizer according to the manufacturer’s standard hagfish Myxine glutinosa, rainbow trout Oncorhynchus protocols. After cleavage and side-chain deprotection, mykiss and Atlantic salmon Salmo salar. Hagfish the crude synthetic peptide was purified on a Vydac cathelicidins were considered as ancient members of the 218TP510 C18 reverse phase-HPLC column (25×1 cm. cathelicidin family (Chang et al, 2005, 2006; Uzzell et al, Separations Group, Hesperia, CA, USA) and eluted at a 2003). flow rate of 2 mL/min by a linear gradient of acetonitrile Recently, we cloned three cDNA sequences coding in 0.1% trifluoroacetic acid in water. Identity of the for elapid cathelicidins from constructed venom gland peptide was confirmed by automated Edman degradation cDNA libraries of Naja atra, Bungarus fasciatus and with a protein sequencer and mass spectrometry analysis. Ophiophagus hannah. The open reading frames of the Fast atom bombardment-mass spectrometry was carried cloned elapid cathelicidins were all composed of 576 bp out on an Autospec-3000 spectrometer, equipped with a and coded for 191 amino acid residue protein precursors. high-field magnet (Lee et al, 2005). The purity of the Each of the deduced elapid cathelicidin has a 22 synthetic peptide was >95% and then used for evaluating amino-acid-residue signal peptide, a conserved cathelin biological activities. domain of 135 amino acid residues and a mature 1.3 Sample preparation for SEM and TEM antimicrobial peptide of 34 amino acid residues. Unlike Overnight culture of E. coli cells (OD =1.2) in 600nm the highly divergent cathelicidins in mammals, the LB medium was diluted 100 times with fresh LB nucleotide and deduced protein sequences of the three medium and gave a total culture volume of 20 mL. Then, cloned elapid cathelicidins were remarkably conserved. the bacteria were continue to culture at 37℃ for OH-CATH, the deduced mature cathelicidin from king additional 4 hours to get mid-logarithmic-phase cobra, showed strong antibacterial activity against organisms (OD =0.7). OH-CATH was added to the 600nm various tested bacteria with minimal inhibitory culture medium to reach a final concentration of 12 g/mL concentration of 1−20 g/mL in the presence of 1% NaCl. and cultured under the conditions as mentioned above. At Meanwhile, the peptide showed no haemolytic activity different time intervals, aliquots were removed and toward human red blood cells even at a high dose of 200 optical density was measured at 600 nm for both g/mL. Elapid cathelicidins might serve as model OH-CATH treated and untreated samples. The samples molecules for the development of antimicrobial drugs prepared for SEM and TEM were as follows: 1) SEM (Zhao et al, 2008c). samples, aliquots of 1 mL culture medium at various No. 2 CHEN Xin-xin et al: Effects of the Antimicrobial Peptide OH-CATH on Escherichia coli 173 culture times were removed to 1.5 mL eppendorf tubes 2008c). Understanding the antibacterial mechanism of and centrifuged at room temperature for 4 minutes (2000 OH-CATH against bacterial pathogens should do a great rpm). After discard the supernatant, 1 mL fresh LB help for the design of more potent antibacterial and better medium were added and vortex thoroughly to wash the specificity for OH-CATH derived peptide(s) against bacterial pellets and centrifuged again at 2000 r/min for 4 microbes while maintain weak cytotoxic to human cells. minutes. After carefully removal of the supernatant, Recently, atomic force microscope (AFM) has been used bacterial cells were chemically pre-fixed with 3.5% to study antimicrobial peptide mechanisms both on glutaraldehyde in 0.1mol/L PBS buffer (pH7.4) at 4℃ model membrane and intact cells. AFM is a powerful overnight. The pre-fixed bacterial cells were collected on imaging tool capable of achieving high resolution images a poly lysine treated microscopy coverslips and washed under physiological conditions and gives 3-D three times with Milli-Q water. The cells on the reconstruction images (Li et al, 2007). Nevertheless， coverslips were post-fixed with 2% osmic acid for 1 hour SEM and TEM are still used to investigate the and subsequently dehydrated with 30%, 50%, 70%, 80%, mechanisms of antimicrobial peptides with microbes 90%, 100% graded ethanol and acetone series. The (Matsuzaki et al, 1997). samples were lyophilized, coated with Pa/Pt and Gram-negative E. coli ATCC 25922 strain was examined by SEM on a Hitachi 300-N instrument. 2) widely used as a control for evaluation of antibacterial TEM samples, aliquots of 1.5 mL culture medium at activity for antibacterial peptides. AFM images of E. coli various culture times were removed to 2 mL eppendorf ATCC 25922 indicated that they showed characteristic tubes and centrifuged at room temperature for 4 minutes rod shape with distinctive peritrichous flagella and pili (2000 r/min). After discard the supernatant, 1.5 mL fresh structure (Li et al, 2007). Previously, we have LB medium were added and vortex thoroughly to wash demonstrated that OH-CATH have a MIC value of 2 the bacterial pellets and centrifuged again at 2000 r/min g/mL in NaCl-free LB media with bacterial inoculation for 4 minutes. After carefully removal of the supernatant, of 5×105 cfu/mL at 37℃ for 16 hours (Zhao et al, bacterial cells were chemically pre-fixed with 3.5% 2008c). In our experiment conditions, one absorption glutaraldehyde in 0.1mol/L PBS buffer (pH7.4) at 4℃ unit at 600 nm approximately equal to 1 ×109 cfu/mL. To overnight. The pre-fixed bacterial cells were post-fixed investigate the interaction of OH-CATH with E. coli with 1% osmic acid for 1 hour and subsequently 25922, a lower OH-CATH concentration of 12 g/mL was dehydrated with 30%, 50%, 70%, 80%, 90%, 100% used to treat E. coli 25922 at a beginning concentration graded acetone series. Epoxy resin (Epon-618) was used of about 7×108 cfu/mL and incubated for 21 hours. It to embed the post-fixed bacterial samples for 12-16 should be noted even the OH-CATH concentration used hours at room temperature. Ultra thin section of the in the present work was 6 times higher than its MIC for embedded samples were prepared by LKB-II Ultracut E. coli 25922 (12 g/mL vs 2 g/mL), but the bacterial instrument and double stained with uranyl acetate and inoculation of E. coli 25922 in the present work was lead citrate. The samples were lyophilized then examined 1000 times higher than that for MIC determination by transmitted electron microscopy on a Hitachi (7×108 cfu/mL vs 5×105 cfu/mL). Obviously, the TEM-1011 instrument. The optical density (OD) at 600 OH-CATH concentration used in the present work was nm was recorded to evaluate the growth of the bacteria. comparatively lower at a beginning bacterial inoculation of 7×108 cfu/mL and allow us to investigate the 2 Results and Discussion time-dependent interaction of OH-CATH against E. coli OH-CATH is a new member of cathelicidin ATCC 25922. E. coli ATCC 25922 growth kinetics of antimicrobial peptides characterized from Elapidae king both OH-CATH treated and un-treated bacterial cells cobra snake. Our previously results indicated that the were shown in Tab. 1. At each determined incubation peptide have a MIC value of 2 g/mL and 8 g/mL against E. intervals, lower antibacterial dosage treated samples coli ATCC 25922 under NaCl-free and in the presence of showed a declined bacterial cell growth compared with 1% NaCl conditions, respectively. Meanwhile, un-treated samples, indicating that OH-CATH do affect OH-CATH showed no hemolytic activity toward human the E. coli ATCC 25922 growth. The determined OD red blood cells up to 200 g/mL, suggestion that this values of OH-CATH treated samples at different molecular might be served as a good model for the incubation intervals also suggested that the OH-CATH at developing of new antibacterial agents (Zhao et al, the fixed concentration of 12 g/mL could not fully kill the 174 Zoological Research Vol. 30 tested bacteria and the bacteria could still grow. from damaged cells. The OH-CATH treated cells showed SEM images suggested that the untreated E. coli cytoplasmic membrane extraction resulted in the cells (Fig. 1A,B) had a smooth surface with average size separation of outer membrane and cytoplasmic of 0.5×2-4 m. The arrows in both Fig. 1A and 1B membrane as indicated by the arrows in Fig. 2D. It indicated the flagella or pili connections between E. coli should be noted that SEM images of bacterial cells after cells. After treated with lower concentration of treated with OH-CATH for 1 or 2 hours showed little OH-CATH for 1 hour, there are litter obviously morphological changes (Fig. 1C, D). Thus, SEM and morphological changes for E. coli cells (Fig. 1C). TEM results of bacterial cells treated with OH-CATH for Pore-forming and morphological changes were observed 1−2 hours suggested that OH-CATH should bind to the for E. coli cells being treated with OH-CATH for 2 hours, bacterial wall and reach a certain concentration then lysis of E. coli cells and release of fluid and debris could aggregate to act in a pore-formation manner to damage be observed (Fig. 1D). After treated with OH-CATH for the cell membrane and release the cell fluid. After treated 6 hours, elongation of cells (>9 µm) and shorten cells with OH-CATH for 6 hours (Fig. 2E, F), many bacterial (<1µm) were clearly observed (Fig. 1E). Meanwhile, cells were damaged by OH-CATH and bleb-like structure collapse of E. coli cell outer membrane and release of existed in many cells (Fig. 2E). Formation of bacterial bacterial fluid could be seen as indicated by the arrows in ghost, which contains intact bacterial envelope, after Fig. 1F. The average pore sizes of bacteria treated with treated with OH-CATH was indicated by the arrow in Fig. OH-CATH were measured to be 100−300 nm (figure not 2E. Bacterial ghost was formed by OH-CATH in a shown). After treated with lower concentration of pore-forming manner and the opening of the bacteria was OH-CATH for 21 hours, the bacteria cells continuously indicated by an arrow in Fig. 2F. After treated with lower grew (Tab. 1) and the morphological changes of the concentration of OH-CATH for 21 hours (Fig. 2G, H), growing cells (Fig. 1G, H) were obviously different from some new growing bacteria cells showed normal the cells untreated with antibacterial peptide (Fig. 1A,B). structure, together with damaged cell debris (Fig. 2G). The most obvious differences were included more The arrows in Fig. 2H indicated the opening of the fiber-like connection among cells as indicated by arrows damaged cells. in Fig 1G, elongation of cell body and cell surface In conclusion, our results indicated that OH-CATH becoming roughness for the growing cells (Fig. 1H). and Gram-negative E. coli ATCC 25922 interactions These images indicated that the cell growth is affected by might happen in three steps. At beginning, OH-CATH the antibacterial peptide. attached to the negatively charged bacterial wall by TEM images suggested that the untreated E. coli positively charged amino acid residues. In the second cells had normal structure (Fig. 1A). The ultra-structure step, with the attached OH-CATH reached a certain of an untreated E. coli cell was shown in Fig. 1B where concentration, the accumulated OH-CATH aggregated outer membrane, periplasm and cytoplasmic membrane and damaged the bacteria membrane in a pore-forming could be seen. After treated with lower concentration of manner, the average pore size of E. coli treated by OH-CATH for 2 hours, some bacterial cells showed OH-CATH was measured to be around 100−300 nm. In morphological changes, like bleb-like structures, were the last step, with the damage of cell permeability, the observed (Fig. 2C, D). The arrow in Fig. 2C indicated contents of the cells were released and the cells that periplasmic and/or cytoplasmic fluid was released eventually collapsed. Tab. 1 Escherichia coli ATCC 25922 growth kinetics at OD 600nm Samples 0 0.5 h 1 h 2 h 6 h 21 h OH-CATH treated 0.708 0.760 0.804 0.852 1.141 1.678 Without OH-CATH 0.708 1.104 1.340 1.548 1.906 2.506 No. 2 CHEN Xin-xin et al: Effects of the Antimicrobial Peptide OH-CATH on Escherichia coli 175 Fig. 1 SEM images of Escherichia coli ATCC 25922 treated with OH-CATH for different times (A−B) At zero time, no OH-CATH added. (C) 1 hour after OH-CATH treated. (D) 2 hours after OH-CATH treated. 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