Ctriporin, a new anti-MRSA peptide from the venom of the scorpion

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Ctriporin, a new anti-MRSA peptide from the venom of the scorpion 1

Chaerilus tricostatus 2

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Running Title: A new anti-MRSA peptide 4

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Zheng Fan*, Luyang Cao*, Yawen He, Jun Hu, Zhiyong Di, Yingliang Wu, 6

Wenxin Li§, Zhijian Cao§ 7

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State Key Laboratory of Virology, College of Life Sciences, Wuhan University, 9

Wuhan, 430072, P. R. China 10

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* These authors contribute equally to this work. 22

§ Corresponding authors. 23

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Address for correspondence: 25

Wenxin Li ([email protected]) and. Zhijian Cao ([email protected]) 26

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 27

Hubei 430072, China Tel: ++86-(0)-27-68752831. Fax: ++86-(0)-27-68756746. 28

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00369-11 AAC Accepts, published online ahead of print on 29 August 2011

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ABSTRACT: Antibiotic-resistant microbes, like methicillin-resistant Staphylococcus 1

aureus, seriously threatened human health. The out break of "super bugs" in recent 2

years once again warned that the development of new antimicrobial agents or 3

resources is emergent. Antimicrobial peptides have an evident bactericidal effect 4

against multidrug-resistant pathogens. Here, a new antimicrobial peptide, Ctriporin, 5

was cloned and characterized from the venom of the scorpion Chaerilus tricostatus, 6

which, so far, has never been explored for toxic peptide resource. The MICs of 7

Ctriporin against Staphylococcus aureus, Bacillus thuringiensis, Bacillus subtilis, 8

Micrococcus luteus, and Candida albicans are 5-20 µg/ml. Meanwhile, its MICs 9

against clinical antibiotic-resistant bacterial strains are 10 µg/ml. Furthermore, the 10

potential for Ctriporin to be used as topical antibiotics for treating staphylococcal skin 11

infections was investigated. External use of the peptide Ctriporin dramatically 12

decreased the bacterial counts and cured the skin infected mice. Additionally, 13

Ctriporin fulfills the antimicrobial efficacy by the bactericidal mechanism of a rapid 14

lysis of cells. Together, all these results exemplify the prospect of developing 15

Ctriporin as new topical antibiotics. 16

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KEYWORDS: Scorpion; Venom peptides; AMPs; MRSA 18


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INTRODUCTION 1

Recently, "superbug" infections are reported frequently worldwide and have gained 2

more and more attention. These so called "superbugs" emerged when antibiotics were 3

over used in the past decades. Unfortunately, methicillin-resistant Staphylococcus 4

aureus (MRSA) is one of the most lethal “superbugs” that is transmitted rapidly all 5

over the world. The first case of MRSA was reported in the United Kingdom in 1961 6

(21, 48). At present, MRSA infections became pandemic in many medical institutions 7

(HA-MRSA) and the community (CA-MRSA) worldwide (17, 29). Moreover, MRSA 8

has been found to colonize or infect livestock and humans exposed to those animals in 9

several countries, and such MRSA has been dubbed livestock-associated 10

methicillin-resistant S. aureus (LA-MRSA) (36). MRSA infections cause many health 11

problems. It has become a primary cause of skin and soft tissue infections among 12

persons without extensive exposure to healthcare settings (46). Such settings and 13

skin-to-skin contact have become a risk factor, which will accelerate the transmission 14

of MRSA in humans (4, 9, 16, 25). MRSA-induced sepsis, endocarditis, pneumonia, 15

soft tissue infections are difficult to treat because of the limited range of antibiotics. 16

Vancomycin is one of the most effective antibiotics against MRSA, but it is associated 17

with many adverse effects (30). Moreover, the over use of vancomycin in treating 18

MRSA infection and the existence of vancomycin-resistant enterococcus has lead to 19

the emergence of vancomycin-intermediate S. aureus (2, 3). In order to prevent more 20

MRSA infections, more effective antimicrobial drugs are needed. 21

Antimicrobial peptides (AMPs), as a part of the ancient and nonspecific innate 22


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immune system, play an important role in defending against invading pathogenic 1

organisms (18, 22, 28). AMPs are typically relatively short (12 to 100 amino acids), 2

positively charged (net charge of +2 to +9) and amphiphilic, which have been isolated 3

from single-celled microorganisms, insects and other invertebrates, plants, amphibians, 4

birds, fish, and mammals, including humans (34, 49). AMPs have a broad spectra of 5

activity against Gram-negative and Gram-positive bacteria, fungi as well as viruses 6

and parasites (26, 39, 45). Moreover, some antimicrobial peptides are identified to 7

have inhibitory activity against antibiotic-resistant bacteria (40). 8

Scorpions are one of the most ancient animals on earth, having survived over 400 9

million years (52). They have been used as a traditional Chinese medicine for more 10

than 2000 years for their outstanding pharmaceutical effects, which include 11

anti-microbial and anti-inflammatory activities (41, 42). It was widely believed that 12

scorpion venom is a mixture of natural bioactive substances (35). At present, some 13

antimicrobial peptides have been found in scorpion venom and hemolymph (8, 20), 14

such as hadrurin (47), scorpine (11), opistoporins, parabutoporin (33), IsCTs (14), 15

pandinins (12), Mucroporin (13), Imcroporin (53) and StCT1 (51), which reveals that 16

scorpion venom is also a rich resource of crude antimicrobial peptides. 17

In our present study, we cloned and identified a new antimicrobial peptide 18

Ctriporin, which is the first bioactive peptide characterized from the venom of the 19

scorpion Chaerilus tricostatus. The mature peptide of Ctriporin was composed of 19 20

amino acid residues with an amidated C-terminus. The synthetic peptide Ctriporin 21

showed potent growth inhibitory activity against standard Gram-positive bacteria and 22


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Candida albicans at low concentration. Moreover, the in vitro treatment of clinically 1

isolated pathogens showed that Ctriporin is able to inhibit antibiotic-resistant 2

pathogens, including methicillin-resistant S. aureus (MRSA), methicillin-resistant 3

coagulase-negative Staphylococcus (MRCNS) and penicillin-resistant Staphylococcus 4

epidermidis (PRSE). We also explored its in vivo antibacterial activity in topical 5

treatment of mouse skin infection model. The result showed that Ctriporin displays 6

reasonable antibacterial activity in vivo using ointment application. Finally, we chose 7

a standard Gram-positive bacteria S. aureus as a model bacteria strain to further 8

explore the antimicrobial mechanism of Ctriporin. Evidences showed that Ctriporin 9

fulfills the antimicrobial efficacy by the bactericidal mechanism of a rapid lysis of 10

cells. All these results suggest that the antimicrobial activity of Ctriporin against 11

Gram-positive bacteria and antibiotic-resistant pathogens makes it a promising 12

candidate for the treatment of skin infection by topical application. 13


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MATERIALS AND METHODS 1

cDNA library construction. The scorpion Chaerilus tricostatus was gathered in 2

Medog Xizang Autonomous Region, China. We cut their glands 2 days after electrical 3

extraction of the venom to provoke their toxin-producing cells into a secretory phase. 4

Their total RNA was extracted using Trizol reagent (Invitrogen). Poly(A)-mRNA was 5

purified by a Poly(A) Tract mRNA Isolation System (Promega). The cDNA library 6

construction was made according to Superscript Plasmid System library construction 7

kit (Gibco/BRL). The cDNAs were cloned into the pSPORT1 plasmid (Gibco/BRL) 8

and introduced into Escherichia coli DH5α cells by transformation. cDNA clones 9

were chose randomly and sequenced to acquire a reliable representation of toxic 10

peptides and proteins in the glands. 11

cDNA sequencing and computer analysis. Positive clones were identified by 12

using an ABI Prism 377XL DNA sequencer with a universal T7 promoter primer. 13

Analysis of the sequence was carried out by using GENERUNR and BLASTX. All 14

homologue sequences of Ctriporin were retrieved from GenBank 15

(http://www.ncbi.nlm.nih.gov/) by the BlastP method. 16

Chemical synthesis and identification. The Ctriporin mature peptide was 17

synthesized by solid-phase synthesis method and was amidated at the C-terminus 18

(More Biotechnology, Ltd., China). Identification of the synthesized peptide and 19

determination of its purity was carried out by RP-HPLC analysis. Agilent 1100 series 20

HPLC equipment and C18 (Agilent Eclipse XDB-C18; 5 µm; 4.6×150 mm) were used 21

Solvents of gradient chromatography were eluent A (0.1% (V/V) trifluoroacetic acid) 22


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and eluent B (90% (V/V) acetonitrile with 0.1% (V/V) trifluoroacetic acid). Average 1

molecular mass of the synthetic Ctriporin peptide was measured by 2

Matrix-assisted-laser-desorption/ ionization time-of-flight mass spectrometry 3

(MALDI-TOF-MS) analysis. Voyager DE STR MALDI-TOF (Applied Biosystems) 4

was used and the matrix was cinnamic acid. 5

Microorganism strains. Standard bacteria strains Bacillus thuringiensis 6

(AB92037), Pseudomonas aeruginosa (AB93066), S. aureus (AB94004), B. subtilis 7

(AB91021), Micrococcus luteus (AB93113), E. coli (AB94012) and standard fungi 8

strain C. albicans (AY93025) were purchased from China Center of Type Culture 9

Collection (CCTCC). 10

Antibiotic-resistant bacterial strains were obtained from the 302nd Military Hospital 11

of Beijing, China, including methicillin-resistant S. aureus (MRSA) P1386 and P1374, 12

methicillin-resistant coagulase-negative Staphylococcus (MRCNS) P1369 and 13

penicillin-resistant S. epidermidis (PRSE) P1389. 14

Antibacterial assay in vitro. 15

MIC. Minimal inhibitory concentrations (MICs) of the peptide against bacterial 16

strains were tested by a liquid growth inhibition assay in 96-well microtiter plates. 17

Bacteria in logarithmic phase were diluted with Luria-Bertani (LB) medium to about 18

104 to 106 CFUs/ml, and fungi in logarithmic phase were diluted with Yeast Extract 19

Peptone Dextrose Medium (YPD). Peptide was diluted to a series of concentrations of 20

400 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3 21

µg/ml, respectively. The bacterial suspension and serial diluted peptides were added to 22


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96-well plates at a ratio of 4:1 in a final volume of 100 µl, and so final concentrations 1

of these peptides were 80 µg/ml, 40 µg/ml, 20 µg/ml, 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 2

1.25 µg/ml, 0.6 µg/ml. The microplates were incubated at 37°C with continuous 3

shaking for 12-16 h. Inhibitory growth was examined by monitoring the absorbance at 4

630 nm with a microplate reader. The concentrations of peptides and commercial 5

antibiotics were all determined by weight. The MIC was determined by optical density. 6

Each concentration reading was repeated three times. All of the above experiments 7

were repeated at least twice. 8

Growth inhibitory effect. Time growth curves of S. aureus, C. albicans and 9

MRSA treated by Ctriporin peptide were measured at 630 nm in each time point. The 10

tested strain added with different concentrations of peptide was cultured in 96-well 11

plates, same as the method mentioned above. Ampicillin, cefotaxime, penicillin, 12

vancomycin and amphoterincin B were chose as controls. 13

Antibacterial assay in vivo. 14

Experimental animals. Adult female BALB/c mice, 6-8 weeks old and weighing 15

17-21 g, were obtained from Hubei Center for Disease Control and Prevention, 16

Wuhan, China. The animals were housed one per cage and maintained on a 12-hour 17

light/dark cycle with access to food and water. All animal studies were approved by 18

the Institutional Animal Care and Use Committee of Wuhan University. 19

Preparation of inoculum....Standard S. aureus (AB94004) was cultured in LB 20

medium at 37°C with continuous shaking. One millilitre AB94004 logarithmic 21

phase broth was quantified and concentrated to 20 µl by phosphate buffered saline 22


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(PBS).. 1

Compound preparation. The synthetic peptide Ctriporin was prepared as an 2

ointment in 0.5% Hydroxypropylcellulose to a final concentration of 5 mg/ml. 3

Mouse skin abrasion and infection model. In order to foster a more vulnerable 4

environment in the mice for infection, mice were injected with two doses of 5

cyclophosphamide at days 4 and 1 before the infection. For the first dose, mice were 6

injected with 150 mg cyclophosphamide per kilogram of the mouse’s body weight 7

(150 mg/kg). The second dose was 100 mg/kg. This treatment reduced peripheral 8

blood neutrophils to <100/ml blood (15). 9

Before the creation of wounds, mice were anesthetized with injections of 20% ethyl 10

carbamate (7 ml/kg mouse body weight) and then shaved on the dorsal surfaces. Skin 11

abrasion wounds were made on the dorsal surfaces of mice using a blade by creating 12

1×1 cm2 scratch area. The abrasion wounds only damaged the stratum corneum and 13

upper-layer of the epidermis but not the dermis. Five minutes after wounding, a 14

pipette tip was used to inoculate 20 µl concentrated suspension containing 107-108 15

CFU of standard S. aureus into each defined wounded area. In each experiment, a 16

group of mice were killed 4 h after infection to control the infectious dose. 17

4 h after infection, the first treatment was introduced. 20 µl 5 mg/ml Ctriporin 18

ointment and placebo ointment were applied to the wound and spread over the area, 19

respectively. 16 h after the first treatment, additional antibiotic applications were 20

made twice daily (in the morning and the evening with an 8 h interval) for a period of 21

4 days. After 4 days of treatment, the mice were killed, and their wound area skin 22


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(approximately 1cm2) was immediately excised. The excised skin was homogenized 1

together with 1 ml of PBS (phosphate buffered saline) in homogenizer. The 2

homogenates were washed once in PBS to decrease the remaining ointment. Suitable 3

dilutions of the homogenates were plated on LB plates to determine the number of 4

living bacteria (CFU). In order to investigate the reproducibility of the infection with 5

S. aureus, three independent experiments that included untreated and placebo-treated 6

groups were performed. 7

Statistical analysis. In each experiment the mean CFU was calculated by using 8

log10-transformed data. Based on the averages of these three experiments, the mean, 9

range, and coefficient of variation were calculated. 10

Histological examination....In order to characterize the histopathology of the model, 11

biopsy specimens were taken after the following treatments: immediately after blade 12

scratching, 4 days after blade scratching, 4 days after inoculation with S. aureus 13

without treatment, with placebo treatment and with Ctriporin treatment. Immediately 14

after the animals were killed, 5-mm punch biopsy specimens of the excised skin were 15

taken and immediately fixed in phosphate-buffered (pH=7.4) formalin (10%). The 16

formalin-fixed biopsy specimens were embedded in paraffin and stained with 17

hematoxylin and eosin. 18

Mechanism investigation 19

Bactericidal assay. Previous studies have suggested that the modes of action of 20

many antibacterial peptides were rapid killing by disrupting the cell membrane of 21

bacteria. Therefore, time-kill curves for Ctriporin and ampicillin against S. aureus 22


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(AB94004) were measured. The bactericidal effects of Ctriporin and ampicillin were 1

tested at one and five times of MIC. Overnight-cultured S. aureus was 1:100 2

transferred to LB and was cultured to exponential phase (OD600 = 0.6). The peptide or 3

ampicillin solution and bacterial suspension were added to a sterilized tube at the 4

volume ratio of 4:1 in a final volume of 2 ml, and the mixture was incubated at 37°C 5

with continuous shaking. At each time point, 200 µl of treated bacterial suspension 6

was transferred to a sterilized 1.5ml tube. After the tube was centrifuged at 1,000 g for 7

5 min, the supernatant was removed and the pellet was resuspended in 200 µl LB 8

medium. Suitable dilutions of the bacterial suspension were placed on LB agar plates 9

and were incubated at 37°C until the viable colonies could be counted. 10

Secondary structure analysis. Secondary structure of the mature peptide Ctriporin 11

was analyzed by Heliquest (http:// heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py) 12

and circular dichroism (CD) assay. CD was performed at room temperature on a 2 mm 13

path length, capped, round optical cell using a Jasco J-810 spectropolarimeter. Spectra 14

were obtained at wavelengths of 190-260 nm. Each spectrum was obtained from 15

averaging three scans taken at a scan rate of 50 nm/min with 2 nm spectral bandwidth. 16

In order to simulate the environment of bacterial cell surface and membrane, 17

trifluoroethanol (TFE) was used as solvent. The synthetic peptide Ctriporin was 18

measured at the concentration of 0.1 mg/ml in water, 30% TFE/H2O or 70% TFE/H2O. 19

The peptide configuration was obtained by analyzing the data with CD deconvolution 20

software. 21

Transmission electron microscope. Overnight-cultured S. aureus was transferred 22


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to LB medium and cultured to the exponential phase. A portion (200 µl) of the peptide 1

solution was added to an 800 µl bacterial suspension, and the mixture was incubated 2

at 37°C with continuous shaking (about 150 rpm/min). After 2 min incubation, the 3

bacterial suspension was immediately centrifuged at 1,000 g for 10 min, and the pellet 4

was washed with 0.1 M PBS several times and then fixed for 1 hour with 2% 5

glutaraldehyde in 0.1 M PBS and followed one hour with 1% osmium tetroxide at 4°C. 6

After fixation, the bacteria were stained by 1% uranyl acetate and then dehydrated by 7

using a series of graded ethyl alcohols (60% for 15 min, 70% for 15 min, 80% for 15 8

min, 90% for 15 min, and 2 changes of 100% for 10 min each). After this, the samples 9

were embedded in epoxy resins and stained by 1% uranyl acetate and lead citrate. The 10

samples were then semi-thin sectioned and ready to view on the Hitachi X650 11

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RESULTS 1

Sequence analysis. After systemic screening of 884 clones from the venom gland 2

cDNA library of the scorpion Chaerilus tricostatus, we got ten positive clones. 3

Sequencing of all these positive clones revealed a new precursor, termed as Ctriporin 4

(Fig. 1). It shares high similarities with caerin-1, caerin-2 and BmKb2 and a number 5

of antimicrobial peptides as shown in Fig. 2. 6

The Ctriporin precursor consists of three parts: 5'UTR, ORF and 3'UTR. The 7

lengths of 5' and 3'UTRs are 86 nt and 71 nt, respectively. At the 3'UTR end of the 8

cDNA, a single aataaa polyadenylation signal is found 16 nt upstream of the poly (A) 9

tail. An ORF of 225 nt encodes a precursor of 75 amino acid residues. 10

Ctriporin has a putative 22-residue signal peptide, identified with the SignalP 3.0 11

program (http://www.cbs.dtu.dk/services/SignalP/), followed by a presumed 12

19-residue mature peptide and an uncommon acidic propeptide at the C terminus. The 13

34-residue propeptide starts with a conserved posttranslational processing signal, 14

Gly-Arg-Arg, at positions 42 to 44. Conventionally, removal of a propeptide with 15

such a processing signal would result in a mature peptide with C-terminal amidation, 16

as described previously (19). The sequence of Ctriporin has been deposited in the 17

GenBank NCBI database (Accession number: JN172934). 18

Chemical synthesis and characterization of Ctriporin. Based on the amino acid 19

sequence analysis of Ctriporin, the presumed 19-residue mature peptide was 20

synthesized by solid phase synthesis method and amidated at the C-terminus. As 21

described previously, Gly followed by either single (Arg), or pairs (Lys-Arg or 22


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Arg-Arg) of, basic residues, are potential cleavage sites for prohormone convertases 1

(27, 43). The presence of multiple copies of peptide sequences flanked by pairs of 2

basic residues is a very common feature of prohormones of insect peptides. Generally, 3

the processing of a prohormone to biologically active peptide with an amidated 4

C-terminus requires the removal of the basic residues to give a peptide intermediate 5

with a C-terminal Gly, which then undergoes oxidation to generate a peptide 6

terminating with an amidated residue (5, 6). C-terminal amidated will increase 7

solubility and potency of the peptide. 8

HPLC analysis showed that the synthetic peptide Ctriporin had a purity of more 9

than 95% (Fig. 3A). Moreover, MALDI-TOF-MS analysis showed that the average 10

molecular mass of the synthetic peptide Ctriporin was 2014.2 Da, corresponding to 11

the m/z signal 2015.2 (Fig. 3B). The average mass tested by MALDI-TOF-MS 12

confirmed the theoretical molecular weight of 2014.5 Da calculated by ProtParam 13

(http://us.expasy.org/tools/protparam.html). 14

Minimal inhibitory concentration. We tested the MICs of the synthetic peptide 15

Ctriporin against standard microorganism strains and antibiotic-resistant pathogens 16

with the microdilution method. As shown in Table 1, Ctriporin peptide can 17

specifically inhibit Gram-positive organisms and fungi, not Gram-negative bacteria. 18

The MICs of Ctriporin against S. aureus (AB94004), B. thuringiensis (AB93066), B. 19

subtilis (AB91021), M. luteus (AB93113), C. albicans (AY93025) were 5 µg/ml, 10 20

µg/ml, 10 µg/ml, 5 µg/ml and 20 µg/ml, respectively. In contrast, E. coli (AB94012) 21

and P. aeruginosa (AB92037) were insensitive to >100 µg/ml Ctriporin peptide. 22


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The MICs of Ctriporin peptide against MRSA, MRCNS and PRSE were tested 1

after determination of their antibiotic resistance. The MICs of Ctriporin peptide 2

against MRSA P1374 and P1386, and MRCNS P1369 were all 10 µg/ml. The MIC of 3

the peptide Ctriporin against penicillin-resistant strain (PRSE) P1389 strain was 10 4

µg/ml. In order to make a direct contrast, the MICs of penicillin G salt, cefotaxime 5

and vancomycin against penicillin-resistant strains and methicillin-resistant strains 6

were all determined. The MIC of penicillin G against the penicillin-resistant strain 7

P1389 was 10,000 µg/ml, which was nearly 3 orders of magnitude higher than that of 8

the peptide Ctriporin. The MICs of cefotaxime against the methicillin-resistant strains 9

were 100 to 400 µg/ml, which was about 10 to 40 times higher than the MICs of 10

Ctriporin peptide (Table 2). Thus, the Ctriporin peptide can not only inhibit the 11

growth of standard Gram-positive bacteria stains and fungi, but also inhibit the growth 12

of clinical isolated MRSA and PRSE strains. Moreover, Ctriporin peptide has a better 13

antibacterial potency than traditional antibiotics in the aspect of inhibiting 14

antibiotic-resistant pathogens. These results indicated that Ctriporin peptide may be a 15

good peptide molecule or compound lead against antibiotic-resistant bacteria, 16

especially MRSA. 17

Growth inhibitory effect. The growth inhibitory effect of Ctriporin to standard 18

microorganism strains and antibiotic-resistant strains was tested by measuring OD600 19

of 5-9 time points with a microplate reader. As shown in Fig. 4A, 5 µg/ml Ctriporin 20

peptide could completely inhibit the growth of S. aureus AB94004 as well as 5 µg/ml 21

penicillin G and 5 µg/ml cefotaxime, while ampicillin can't effectively inhibit its 22


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growth 7 hours after treatment in the same concentration. When the concentration of 1

Ctriporin peptide was decreased to 3.5 µg/ml, S. aureus growth could be inhibited for 2

the first 7 h, but when the concentration decreased to 2.5 µg/ml, S. aureus growth was 3

inhibited in the first 4 hours. This result suggested that the inhibitory effect of 4

Ctriporin against S. aureus was concentration dependent. For C. albicans, its effect 5

was similar (Fig. 4B). 6

In addition, 500 µg/ml penicillin or 200 µg/ml cefotaxime failed to inhibit the 7

growth of MRSA P1374 strain (Fig. 4C). In contrast, 10 µg/ml Ctriporin peptide could 8

completely inhibit the growth of MRSA P1374 strain, although when the 9

concentration of the peptide Ctriporin was decreased to 5 µg/ml, it only slightly 10

inhibited the bacterial growth. As shown in Fig. 4D, 500 µg/ml penicillin G failed to 11

inhibit the growth of PRSE strain P1389, while both 10 µg/ml and 5 µg/ml Ctriporin 12

peptide can effectively inhibit the growth of PRSE P1389 within 8 hours. But when 13

the concentration of the peptide Ctriporin was decreased to 2.5 µg/ml, Ctriporin 14

nearly lost its antibacterial activity to PRSE strain P1389. Therefore, the inhibitory 15

effect of Ctriporin peptide against antibiotic-resistant bacteria was also concentration 16

dependent. 17

CFU statistics. The number of CFU recoverable from the wound 4 h after 18

application of 107-108 CFU of S. aureus was 7.62 ± 0.14 log10. There was no 19

significant difference (P = 1.0) in the numbers of CFU per wound as well as 4 days of 20

untreated (7.38 ± 0.16 log10) versus 4 days of placebo treated (7.36 ± 0.15 log10). But 21

the comparison of placebo treatment (7.36 ± 0.15 log10) and Ctriporin (4.48 ± 0.16 22


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log10) treatment revealed a great reduction in the number of CFU per wound (Fig. 5), 1

which was statistically significant (P <0.001) (Table 3). This result indicates that 2

Ctriporin peptide as a topical antibiotic treatment is effective in treating the mice skin 3

S. aureus infection. 4

Wound morphological observation. Wound morphologies were observed daily. 5

As shown in Fig. 6, one day after infection, the skin treated with Ctriporin peptide 6

scabbed over, and got dry and crusty, while the skin treated with placebo and no 7

treatment was moist, soft and red. Four days after infection, the scabs fell off and no 8

visible abrasion was observed in the Ctriporin treated skin, while the placebo treated 9

skin was still red swollen and wet, and the untreated skin appeared effusion and 10

ulceration with cave formation. 11

Histological examination. After 4 days treatment for infection, biopsy specimens 12

were dissected immediately, and then fixed in formalin and embedded in paraffin. The 13

biopsy specimens were stained with hematoxylin and eosin, and then sectioned and 14

observed under an optical microscope. As shown in Fig. 7B, blade scratched skin had 15

most of the epidermis removed when compared with the normal skin (Fig. 7A), 16

although a single layer of epidermal cells or scattered epidermal cells remained. 17

Except for the missing epidermis, the skin appeared normal. Four days after blade 18

scratching, the layer of epidermal cells were restored and the skin recovered (Fig. 7C). 19

Four days after infection, the epidermis nearly disappeared and the dermis was badly 20

damaged in the placebo treated group (Fig. 7E) and untreated group (Fig. 7F). 21

Moreover, the presence of bacteria was evident on the surface of the ulcer in both the 22


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untreated and placebo treated groups. But it was very interesting that, in the Ctriporin 1

treated group, the skin had basically recovered and a relatively thin epidermis and 2

normal dermis were formed (Fig. 7D). A thin layer of corneum was also formed on the 3

surface of the Ctriporin treated group skin. 4

Bactericidal assay. The bactericidal assay results showed that the bactericidal 5

process started from the moment that the bacteria were mixed with Ctriporin peptide. 6

As shown in Fig. 8, the visible colonies of 1×MIC Ctriporin treated bacteria 7

observably decreased to a platform and the colony number of 5×MIC Ctriporin treated 8

bacteria decreased steeply to a much lower platform in 5 minutes. Thus, we can 9

conclude that the killing rate positively corresponded with the concentration of 10

Ctriporin peptide. In contrast, the 5×MIC of ampicillin sodium (40 µg/ml) made a 11

gradual decrease in survival rate, which was consistent with its molecular mechanism 12

of inhibiting the synthesis of the bacterial wall. This result suggested that Ctriporin 13

peptide exerted a rapid killing effect on microorganisms, which was probably the 14

reason why it can effectively inhibit the growth of microorganisms. 15

Secondary structure analysis of Ctriporin. The secondary structure of Ctriporin 16

peptide predicted online was a typical α-helix (Fig. 9A). The helical wheel of 17

Ctriporin peptide was divided into two parts: one part was the hydrophobic face, and 18

the other was the hydrophilic face where there are two positively charged lysines. This 19

result suggested that the peptide Ctriporin was an amphipathic peptide molecule. 20

Furthermore, the result of CD spectra analysis also confirmed that Ctriporin peptide 21

was a typical α-helix molecule. As shown in Fig. 9B, Ctriporin showed high 22


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percentages of α-helix (>90%) in the structure promoting solution, 70% and 30% 1

aqueous TFE. However, Ctriporin peptide displayed the secondary structure of 2

random coil in water. All these results indicate that Ctriporin peptide is a positively 3

charged molecule which can form amphipathic helical structure under appropriate 4

environmental conditions. However, it was well known that proline and glycine 5

residues may break up alpha helixes. Whether the peptide Ctriporin with proline and 6

glycine residues can form the helical structure under appropriate environmental 7

conditions needed to be clarified by determining the high structure of the peptide 8

Ctriporin. Probably the helix of Ctriporin peptide may start after proline or glycine 9

residues. 10

Transmission electron microscopy. S. aureus cells treated with Ctriporin peptide 11

were observed with a transmission electron microscope to present single bacteria 12

changes. As shown in Fig. 10C and Fig. 10D, some pores and cracks were formed on 13

the surface of S. aureus cell walls which have been divorced from the membranes. 14

Moreover, the cracking tended to occur on dividing cells which have produced a 15

relatively larger distortion on the cell walls (Fig. 10E). More importantly, some 16

bacterial cells were completely broken into fragments, leading to the leakage of cell 17

contents (Fig. 10F). All these results were direct evidence supporting the idea that 18

Ctriporin peptide treating caused a fragile cell wall that bursts, and then killed the 19

bacteria. 20

The results of bactericidal assay, secondary structure analysis and transmission 21

electron microscopy provided clear evidence that the bacterial cell walls were broken 22


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immediately after Ctriporin treatment and the inhibitory effect of Ctriporin on bacteria 1

was through the action mode of rapid killing. 2


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DISCUSSION 1

AMPs activation is one of the primary mechanisms used by human skin in the early 2

stages of immune defense (50). Many cell types that permanently reside in skin 3

produce AMPs, including keratinocytes, sebocytes, eccrine glands, and mast cells (7, 4

31). AMPs are important for skin health, less expression of them may result in some 5

skin diseases like atopic dermatitis (37), skin injury and infection (10, 38). AMPs 6

appear to form a chemical shield on the surface of the skin and protect human skin 7

from the infection of invading pathogens. Damaged skin tends to be more vulnerable 8

to pathogens, especially MRSA. 9

Just like AMPs found in mammal skin, AMPs found in scorpion venom are also 10

important and diverse. Since the first two antimicrobial peptides Hadrurin (47) and 11

Scorpine (11) were isolated from the scorpion Hadrurus aztecus and the scorpion 12

Pandinus imperator, some AMPs were isolated and identified from the venoms of 13

different scorpion species. The venom gland cDNA library of the scorpion Chaerilus 14

tricostatus was constructed first. Further, Ctriporin was the first antimicrobial peptide 15

found in this scorpion. It consists of 19 amino acids with a net positive charge of 2, 16

and the amino acid sequence has about 36.8–47% similarity to that of BmKb2 (32), 17

Imcroporin (53) and Mucroporin (13), while the MIC of Ctriporin peptide against S. 18

aureus is 5 µg/ml, which was about 3-5 folds lower than that of BmKb2, Imcroporin 19

and Mucroporin. Moreover, the MIC against MRSA was even 5-10 folds lower. So, it 20

provides us a new template for anti-infective drug design and gives us a clue that 21

Ctriporin has the potential to be designed as an effective antimicrobial agent for some 22


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infection disease. 1

Since there are no effective treatment strategies for some serious skin infections, 2

especially MRSA infection, our study focused on the effect of external use of 3

Ctriporin peptide. Results suggest that Ctriporin may become a potential candidate for 4

treating skin infection. In vitro, Ctriporin can effectively inhibit Gram-positive 5

bacteria including clinically isolated antibiotic-resistant pathogens (MRSA, MRCNS, 6

etc). Besides, it also has antifungal activity against C. albicans. A test for the 7

efficiency of external use of peptide Ctriporin was carried out in vivo. The results 8

show that Ctriporin topical treatment can effectively protect skin from infection in 9

mouse skin S. aureus infection model. Ctriporin treatment with 0.1 mg per wound 10

resulted in a greater decrease in bacterial counts of S. aureus from untreated controls, 11

3.38log10 decreased on average. Similarly, both morphological observation and 12

histological examination show that the peptide Ctriporin treated skin was back to 13

normal. Moreover, Ctriporin has advantages in some aspects when compared with 14

Pexiganan (MSI-78) and Omiganan Pentahydrochloride (MBI 226), which have been 15

employed as topical agents in gel formulations. Ctriporin is more effective in 16

inhibiting C. albicans. The MIC of Pexiganan and Omiganan against C. albicans is 17

64- >512 µg/ml (24) and 32-128 µg/ml (44), respectively, which were about 3-5 fold 18

higher than that of Ctriporin by average. Besides, the antibacterial activity of 19

Ctriporin against MRSA is also better than that of Pexiganan (MIC, 16-64 µg/ml) (24) 20

and Omiganan (MIC, 16-32 µg/ml) (23). So, Ctriporin may be complementary to 21

these developed topical antibiotics in some functions. 22


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Unlike conventional antibiotics, which generally target a metabolic enzyme and 1

may selectively induce resistance in micro-organisms, AMPs kill microbes mainly by 2

membrane-targeting pore-forming mechanisms, a mechanism that is inherently more 3

difficult for microbes to circumvent by developing resistance (26). The relatively 4

short length of AMPs makes them reasonably easy to synthesize, which has lead to a 5

vast number of studies and clinical trails being made (1, 21, 39). All of these suggest 6

that the broad spectrum antibacterial activity and low risk of drug resistance may 7

enable antimicrobial peptides to be one of the alternatives to traditional antibiotics. 8

In conclusion, we discussed here that Ctriporin, being a new member of 9

antimicrobial peptide family, has shown a strong antimicrobial activity, including 10

Gram-positive bacteria, fungi and antibiotic-resistant pathogens (MRSA, MRCNS and 11

PRSE). It is a new template for anti-infective drug design. It rapidly killed the bacteria 12

and effectively cured skin infected mice by external use. Therefore, we consider that 13

Ctriporin, for its high activity and therapeutic potential in skin infection, may have a 14

good prospect in clinical application. 15


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ACKNOWLEDGEMENT 1

Thank Ruiming Zhao from University of Chicago for the language correction of 2

this article. This work was supported by grants from NSFC (No. 31071942), the 3

China Specific Project for Developing New Drugs (Nos. 2009ZX09103-612 and 4

2008ZX10001-015-9), and the National Basic Research Program of China (Nos. 5

2010CB529800 and 2010CB530100), and the Fundamental Research Funds for the 6

Central Universities (No. 1102001). 7


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Table 1. Antibacterial effects of Ctriporin peptide against standard strians 1

MIC (µg/ml) Strains

Ctriporin Penicillin Cefotaxime Amphoterincin B

Bacillus thuringiensis

AB92037 10 12.5 12.5 ND

Bacillus subtilis

AB91021 10 50 50 ND

Staphylococcus aureus

AB94004 5 6.25 3 ND

Micrococcus luteus

AB93113 5 25 3 ND

Escherichia coli

AB94012 >100 ND 3 ND

Pseudomonas aeruginosa

AB93066 >100 ND 12.5 ND

Candida albicans

AY93025 20 ND ND 1.5

ND, not determined 2


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Table 2. Antibacterial effects of Ctriporin peptide against clinical isolates 1

MIC (µg/ml) Strains

Ctriporin Vancomycin Cefotaxime Penicillin

Methicillin-resistant S.aureus

P1386 10 4 100 10000

P1374 10 8 400 4000

P1369 10 8 400 20000

Penicillin-resistant S. epidermidis

P1389 10 8 2 10000

Methicillin-sensitive S.aureus

AB94004 5 3 3 6.25

All strains were clinical isolates obtained from the 302nd

military hospital, Beijing, China. 2


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Table 3. Bacterial counts obtained in the various treatment groups 1

Mean bacterial count (Log10 CFU/wound)±SD

(no. of wounds)

Groups

Staphylococcus aureus(AB94004)

Inoculum size 7.66±0.09 (32)

4 h postinfection 7.62±0.14 (8)

Untreated (day 4) 7.38±0.16 (8)

Placebo treated (day 4) 7.36±0.15 (8)

Ctriporin treated (day 4) 4.48±0.16 (8)

Ctriporin≠Placebo (P < 0.001); Ctriporin≠Untreated (P < 0.001); Untreated = placebo (P = 1.00). 2


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Figure legends: 1

Fig. 1. The precursor nucleotide sequences and the deduced amino acid sequences of 2

Ctriporin (Accession number JN172934). The deduced amino acid residues are 3

shown below the corresponding nucleotide sequences. The signal peptide 4

residues were underlined. The mature peptide residues were highlighted with 5

gray color. The C-propeptide residues were not marked. The potential 6

polyadenylation signal aataaa was double underlined. 7

Fig. 2. Sequence alignments of Ctriporin and its related peptides. The sequence 8

alignments of Ctriporin with other antimicrobial peptides were carried out by 9

DNAMAN. The residues shaded in dark blue are highly conserved sites 10

(100%), the residues in pink and light green are less conserved sites (≥75% 11

and ≥50%), and the residues without background color are highly varied sites. 12

Identity%, the percentage of sequence identity relative to Ctriporin. 13

Fig. 3. HPLC profile and mass spectrum of the synthetic Ctriporin peptide. (A) HPLC 14

profile of the synthetic Ctriporin peptide. The fraction containing the synthetic 15

Ctriporin peptide was indicated. (B) Mass spectrum of the synthetic Ctriporin 16

peptide measured by MALDI-TOF-MS. Measured molecular weight 17

corresponding to the m/z signal (2015.2) was 2014.2 Da and the theoretical 18

calculated molecular weight was 2014.5 Da. 19

Fig. 4. Time growth curves of S. aureus and C. albicans treated by Ctriporin peptide. 20

The growth was measured at 630nm. (A) Comparison of Ctriporin and 21

antibiotics activities against S. aureus AB94004. (B) Comparison of Ctriporin 22


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and antibiotics activities against C. albicans AY93025. (C) Comparison of 1

Ctriporin and antibiotics activities against clinical isolated MRSA P1374. (D) 2

Comparison of Ctriporin and antibiotics activities against clinical isolated 3

PRSE P1389. 4

Fig. 5. Blade scratched mice infected with S. aureus AB94004. The number of 5

bacteria (CFU) extracted from each mouse is represented by the symbol for 6

the corresponding experimental group. The median value of the data for each 7

group is shown as a horizontal bar. The number of mice in each experimental 8

group was as follows: 4 h postinfection, n = 8; untreated, n = 8; placebo, n = 8; 9

Ctriporin, n = 8. 10

Fig. 6. Antibacterial effects of the peptide Ctriporin in vivo. Mice wound 11

morphologies were observed after treated by Ctriporin or placebo and 12

untreated in day 1 and 4. 13

Fig. 7. Histological appearance of (A) normal dorsal skin of mice; (B) immediate after 14

blade scratched skin; (C) 4 days after blade scratched skin; (D) 4 days after S. 15

aureus infected skin with Ctriporin treatment; (E) 4 days after S. aureus 16

infected skin with placebo treatment; (F) 4 days after S. aureus infected skin 17

with no treatment. Biopsy specimens were taken immediately after the 18

termination of the experiment, fixed in formalin, and embedded in paraffin. 19

The biopsy specimens were stained with hematoxylin and eosin. Infected 20

lesions are present in boxes. Numbered arrows indicate in the following: 1, 21

corneum; 2, epidermis; 3, dermis; 4, muscular layer; 5, bacteria. 22


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Fig. 8. Killing assay. Killing assay was conducted with determining the count of the 1

surviving bacteria without the supernatant. Set point in the picture meant the 2

initial bacterial count: the 0 minute was defined as the time of the first sample 3

collection, which was immediately after mixing bacteria and Ctriporin, and the 4

other samples were collected at 5 min, 15 min, 30 min and 60 min after 0 min. 5

All the counts were the average of three dishes. The experiment was repeated 6

and showed the same trend. 7

Fig. 9. Secondary structure analysis of Ctriporin peptide. (A) Helical wheel diagram 8

of Ctriporin peptide by Heliquest method. Representation of Ctriporin mature 9

peptide as helical wheel showed the hydrophilic face and hydrophobic face. (B) 10

Circular dichroism spectra of 0.1 mg/ml Ctriporin in water, 30% and 70% 11

aqueous TFE. 12

Fig. 10. The transmission electron microscopy images of S. aureus treated by 13

Ctriporin peptide. (A, B) Negative control; (C, D, E, F) Ten minutes after 14

Ctriporin treatment. Numbered arrows indicate in the following: 1, pores on 15

cells; 2, cracks on cell walls; 3, cell lysis. 16


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Ctriporin, a new anti-MRSA peptide from the venom of the scorpion

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