Induction, Purification and Characterization of An Antibacterial Peptide Scolopendrin I From The Venom of Centipede Scolopendra Subspinipes Mutilans | High Performance Liquid Chromatography | Physical Sciences

Indian Journal of Biochemistry & Biophysics Vol. 43, April 2006, pp 88-93

Induction, purification and characterization of an antibacterial peptide scolopendrin I from the venom of centipede Scolopendra subspinipes mutilans Ren Wenhua1, Zhang Shuangquan1*, Song Daxiang2, Zhou Kaiya2 and Yang Guang2 1

Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210097, P. R. China 2 Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210097, P. R. China Received 15 June 2005; revised 17 November 2005 The crude venom of the centipede Scolopendra subspinipes mutilans, injected with Escherichia coli K12D31 for 3-4 days showed broad-spectrum antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. It showed good antibacterial activity against E. coli K12D31 at different temperatures, pH, and ionic strengths. The crude venom was heated at 100°C for 30 min, centrifuged at 10,000 rpm for 30 min at 4°C and the supernatants were obtained, from which an antibacterial fraction having a molecular mass of 3000-5000 Da, was further separated by ultrafiltration. A homogeneous antibacterial peptide named scolopendrin I, having a molecular mass of 4,498 Da, was isolated using cation-exchange chromatography and two steps of reverse-phase high performance liquid chromatography (RP-HPLC). Scolopendrin I did not show any hemolytic and agglutination activities at the concentration below 30 µM.

Keywords: Centipede, Scolopendra subspinipes mutilans, antibacterial peptide, scolopendrin I, venom, E. coli K12D31, induction, hemolytic activity, agglutination activity

The centipede Scolopendra subspinipes mutilans is widely used in traditional medicine for the treatment of neural, respiratory and cardiovascular diseases in many Asian countries1. However, only a few reports are available about purification and characterization of proteins/peptides from the centipede. A highmolecular mass acidic and heat-labile cardiotoxic protein (toxin S) was isolated from the venom of S. s. dehaani2. Saxiphilin, a transferrin was purified from the centipede Ethmostigmus rubripes could detect the paralytic shellfish toxins3. Also, a novel serine protease scolonase, composed of 277 amino acids was characterized from S. s. mutilans4. In this study, an antibacterial peptide tentatively named scolopendrin I was purified from the crude venom of S. s. mutilans when injected with E. coli K12D31. The molecular mass, antibacterial, hemolytic and agglutination activities of scolopendrin I were preliminarily investigated. Materials and Methods Adult centipedes (Scolopendra subspinipes mutilans) each weighing around 3 g were procured __________ *To whom correspondence should be addressed Tel.: 86-25-83598216; E-mail: [email protected] Abbreviations: poly IC, polyinosinic-polycytidylic acid; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography; RP-HPLC reverse-phase HPLC, PBS, phosphate-buffered saline, MIC, minimal inhibitory concentration

from Chuzhou, Anhui Province, P. R. China. Grampositive strains Bacillus subtilis ACCC 11062, Staphylococcus aureus CFCC 1117 and Streptococcus pyogenes CVCC 594; Gram-negative strains Escherichia coli ACCC 12069, Pseudomonas aeruginosa CMCC (B) 10101, Shigella dysenteriae CMCC (B) 51832 and Proteus mirabilis CMCC (B) 49005; and fungal strains Saccharomyces cerevisiae ACCC 2032, Aspergillus niger ACCC 30005 and Mucor bacilliformis AS 3.3420, used in the study were provided by the China General Microbiological Culture Collection Center, Beijing, China. Induction of antibacterial substances, extraction of crude venom and assay of antibacterial activity

Antibacterial molecules were induced as described5. Briefly, each centipede was injected with approx. 106 viable, log-phase E. coli K12D31 150 µL, physiological saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2) 50 µL and poly IC 50 µL. After 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7 and 8 day, the crude venom from these ten groups of centipede (5 animals in each group) was collected by an electrical milking procedure6 and centrifuged at 10,000 rpm for 30 min at 4°C. The supernatants were lyophilized and stored at -70°C in ultracold freezer for further assay. Antibacterial activity of crude venom was determined by measuring zones of growth inhibition in thin LB-agar plates with E. coli K12D31

WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE

(welldiffusion assay)7. Each well of 1 mm in diameter was loaded with 5 µL venom and then incubated at 37°C for 12 hr. The minimum inhibitory concentration (MIC) of crude venom against microorganisms was tested as described8. Unlike bacteria, fungi were inoculated in potato-dextrose at 25°C. The optical density at 620 nm (OD620) was measured. Effect of different temperatures, pH, and ionic strengths on antibacterial activity of crude venom Effect of temperature

The lyophilized powder of crude venom from centipede induced by E. coli for 4 days was dissolved in PBS (0.02 M, pH 5.0) (final conc. 1 µg/µL) and then incubated at ten different temperatures: -20, 4, 15, 25, 37, 50, 70, 90, 100 and 121°C for 30 min. Effect of pH

The lyophilized powder of crude venom from centipede induced by E. coli for 4 days was dissolved in disodium hydrogen phosphate-citric acid buffer (final conc. 1 µg/µL) at pH 2.2, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, for 48 hr. Effect of ion strength

The lyophilized powder of crude venom from centipede induced by E. coli for 4 days was dissolved in pH 6.0 and pH 8.0 disodium hydrogen phosphatecitric acid buffer (0.2, 0.02 and 0.002 M) (final conc. 1 µg/µL), for 48 hr. Antibacterial activity of all the above crude venom samples i.e., at different temperatures, pH, and ionic strengths, was tested by 96-well microtiter plate. Each well contained 40 µL LB medium culture, with approx. 1 × 105 E. coli K12 D31/mL and 5 µL of the crude venom samples. Microbial growth was monitored by optical density (OD) at 492 nm, after incubation for 18 hr at 37°C. The controls were performed using bacteria alone, in the presence of the same buffer. Purification of scolopendrin I

The crude venom, which showed activity against E. coli K12 D31, was dissolved in ultrapure water (Arium® 611 water ultrapurifier, Sartorius, Germany), heated at 100°C for 30 min in water bath and centrifuged at 10,000 rpm for 30 min at 4°C. The supernatants after concentration and ultrafiltration (using Vivaspin 0.5 ml concentrator Vivascience, Sartorius AG, Germany) were divided into three parts with bases on the molecular mass of 5,000 Da, respectively and their antibacterial activity was assayed7. The ultrafiltrate of

89

3,000-5,000 Da was separated by cation-exchange chromatography on TSK-gel SP-5PW column (Toso, Japan), initially equilibrated with 0.05 M ammonium acetate buffer, pH 5.1. The column was eluted with a linear gradient of 0-80% of 1 M ammonium acetate buffer, pH 5.1 over 30 min, at a flow rate of 0.5 ml/min. The fraction that showed antibacterial activity was collected and lyophilized. The lyophilized powder was diluted with 1% TFA and purified by RP-HPLC using a Hypersil BDS C18 column (4.6 × 150 mm, 5 µm, Dalian Elite Analytical Instruments Co. Ltd., DEAIC), equilibrated with 1% TFA. The column was eluted with a linear 0-50% gradient of acetonitrile in acidified water over 30 min, at a flow rate of 0.7 ml/min and the active fraction was collected and lyophilized. The active fraction was further purified by a RP-HPLC using a Kromasil KR60 5CN column (4.6 ×150 mm, EKA Chemicals AB, Sweden), equilibrated with 1% TFA and eluted with a linear 0-5% gradient of acetonitrile in acidified water, at a flow rate of 0.7 ml/min. All chromatography steps were carried out at room temperature on a HPLC detection system (Hewlett Packard 1050). The column eluent was monitored for absorbance at 280 nm. The fractions with one individual peak were hand-collected and lyophilized under vacuum. Only one peak was available in the final purification step (Fig. 7), which was collected, lyophilized and tentatively named scolopendrin I. At all the purification steps, antibacterial activity of the products was determined by measuring growth inhibition zones (in mm) on thin agarose plates, inoculated with E. coli K12 D31. Luria agarose (pH 6.4) was mixed with 60 µL of 10-4 dilution (approx. 2 × 105 cells) of log-growth phase of E. coli K12D31. The lyophilized powder of the product obtained at each step was dissolved in sterile saline solution with a concentration of 100 µg/mL. The 10 µL solution was filled in wells and the plates were incubated at 37°C for 24 hr. Synthetic cecropin B (Sigma) and sterile saline solution were used as positive and negative controls, respectively. Total and specific antibacterial activities of the products from each step were calculated. One unit of antibacterial factor activity was defined to be equivalent to the activity of 0.1 µg of synthetic cecropin B9. Tricine-SDS-PAGE

Tricine-SDS-PAGE of scolopendrin I was carried out as described earlier10. Gel was cut vertically down the middle after electrophoresis. One half was stained

90

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, APRIL 2006

with Coomassie blue R250 and the other half was overlaid with media containing viable cells of E. coli K12D31 to display the antibacterial activity zone. Molecular mass

Molecular mass of scolopendrin I was determined using MALDI-TOF-MS with a Bruker ReflexTM (Bremen, Germany) by the National Center of Biomedical Analysis, the Chinese Academy of Military Medical Sciences, Beijing, China. Hemolytic and agglutination activities of scolopendrin I

Scolopendrin I dissolved in PBS buffer (50 mM sodium phosphate buffer, 150 mM NaCl, pH 7.2) (final conc. was 2.5, 5, 10, 15, 20, 25 and 30 µM) was mixed with the same volume of mouse erythrocytes in the same buffer (final 1%, v/v). The negative control (0% hemolysis) consisted of erythrocytes in PBS buffer, whereas the positive control (100% hemolysis) consisted of the erythrocytes, mixed with honeybee venom melittin, dissolved in PBS buffer. All samples, including dissolved scolopendrin I and the controls, were incubated with gentle shaking (50 rpm) at 37°C for 1 hr, placed on ice and centrifuged at 3,000 rpm for 10 min at 4°C to obtain the supernatants. The hemolytic activity of scolopendrin I was determined by measuring the absorbance of the supernatant at 546 nm. The agglutination activity was assayed visually. A negative control was set as above, but the positive control used was phytohaemagglutinin, not melittin. Results and Discussion Antimicrobial activity and physicochemical properties of crude venom

Being a soil animal, S. subspinipes is inevitably exposed to microorganisms, such as bacteria and fungi,

etc. Thus, it is anticipated to produce some antibacterial substances for predation and self-protection. After S. subspinipes was injected with E. coli K12D31 and poly IC, its venom showed antibacterial activity. Although no antibacterial activity was detected in the crude venom at 0.5 day, after 1 day, diameter of the inhibition zone increased to 5.2 cm and thereafter, the activity increased gradually to peak after 3-4 days. Thus, centipedes injected for 4 days were used for the isolation of antibacterial peptide. The activity almost remained stable from the 5th to 8th day (Fig. 1). The crude venom exhibited antimicrobial activity against Gram-positive bacteria B. subtilis, S. aureus and S. pyogenes, Gram-negative bacteria E. coli, P. aeruginosa and S. dysenteriae and the fungi S. cerevisiae, A. niger and M. bacilliformis (Table 1). Thus, the activity was relatively broad spectrum, though, no obvious activity was found against P. mirabilis. Compared with the controls, the venom sample (from centipede induced by E. coli for 4 days) treated at different temperatures, pH and ionic strengths showed Table 1―Antimicrobial activity of crude venom of Scolopendra subspinipes mutilans Minimum inhibitory conc. (µg/mL) Bacillus subtilis ACCC 11062 Staphylococcus aureus CFCC 1117 Streptococcus pyogenes CVCC 594 Escherichia coli ACCC 12069 Pseudomonas aeruginosa CMCC (B) 10101 Shigella dysenteriae CMCC (B) 51832 Proteus mirabilis CMCC (B) 49005 Saccharomyces cerevisiae ACCC 2032 Aspergillus niger ACCC 30005 Mucor bacilliformis AS 3.3420

4.1 5.6 7.2 3.3 7.2 4.1 NA 14.4 5.6 14.4

NA, no activity

Fig. 1―Antibacterial activity of crude venom of Scolopendra subspinipes mutilans, injected with E. coli K12D31, as indicated by the diameter of inhibition zone [Values are averaged from duplicated experiments]

Fig. 2―Effect of temperature on crude venom of S. s. mutilans [OD values at 492 nm of E. coli K12D31 incubated with crude venom at ten different temperatures for 30 min]

WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE

lower OD values, indicating the antibacterial activity had thermal (Fig. 2), pH (Fig. 3) and ionic strength (Fig. 4) stability. The thermal stability, insensitivity to changes of pH and ionic strength of crude venom showed a great similarity to insect antibacterial peptides11. Purification of scolopendrin I

From the crude venom, without being heated up to 100°C for 30 min, five fractions, based on the molecular mass i.e., 30,000 Da were separated by ultrafiltration. All the fractions showed antibacterial activity, suggesting that centipede could produce peptides/proteins with various molecular masses after induction. When the crude venom was heated up to 100°C for 30 min, total amount of protein decreased significantly from 1364.11 mg to 298.32 mg, while 44.88% antibacterial activity (i.e. purification yield9) was retained. The specific antibacterial activity, however, increased significantly after the heat-treatment (Table 2). Thus, heat-treatment was efficient to remove unwanted proteins at the beginning of the purification procedure. Furthermore, when the crude venom was

Fig. 3―Effect of pH on the crude venom of S. s. mutilans [OD values at 492 nm of E. coli K12D31 incubated with the crude venom at different pH for 48 hr]

91

incubated at 100°C for 30 min, the fraction 3,0005,000 Da showed highest antibacterial activity, compared to other fractions. Therefore, heat-treatment was chosen as the first step for the purification of scolopendrin I, and then, ultrafiltrate of 3,000-5,000 Da was collected and fractionated on TSK-gel SP5PW column. The yield and the antibacterial activity against E. coli K12 D31 of the products from different purification steps are given in Table 2.

Fig. 4―Effect of ionic strength on the crude venom of S. s. mutilans [OD values at 492 nm of E. coli K12 D31 incubated with crude venom at pH 6.0 and pH 8.0 disodium hydrogen phosphatecitric acid buffer for 48 hr]

Fig. 5―Cation-exchange chromatography of the ultrafiltrate of 3,000-5,000 Da from crude venom on a TSK-GEL SP-5PW column [The height of bars is proportional to antibacterial activity of the fractions against E. coli. The solid line represents absorbance of fractions at 280 nm and broken line represents a gradient of 1 M ammonium acetate]

Table 2―Antibactericidal activity against E. coli K12 D31 and yield of products from each purification step for scolopendrin I [Synthetic cecropin B was used as positive control and sterile saline solution as the negative control. One unit of antibacterial factor activity was defined to be equivalent to the activity of 0.1 µg of synthetic cecropin B] Crude venom Heated at 100°C for 30 min Ultrafiltrate (3000-5000 Da) Cation-exchange (using HPLC P-5PW) RP-HPLC (using BDS C 18) RP-HPLC (using KR-60 5CN)

Total protein (mg) 1364.11 298.32 35.65 1.21

Total activity (KU) 82.42 36.99 7.028 5.603

Specific activity (KU/mg) 0.060 0.124 0.197 4.631

Purification yield (%) 100 44.88 8.53 6.79

0.78 0.77

2.711 2.695

3.475 3.500

3.29 3.27

92

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, APRIL 2006

Nucleic acid, which carries negative charge, was removed from the sample by the cation-exchange chromatography. Three peaks A, B and C, all having antibacterial activity, were obtained. As the activity of fraction C was most significant (Fig. 5), it was collected, purified and separated by RP-HPLC on Hypersil BDS C18 column, as shown in Fig. 6. The highest peak inhibited the growth of E. coli. K12D31,

and was further purified by RP-HPLC using Kromasil KR-60 5CN (Fig. 7), pooled and lyophilized. The purified fraction was tentatively named scolopendrin I. Its molecular mass as determined by MALDI-TOF mass spectrometry was 4,498 Da (Fig. 8). Tricine-SDS-PAGE showed only one band with molecular mass in the range of 4.0-6.0 kDa, suggesting scolopendrin I is a small polypeptide (Fig. 9A). It exhibited an antibacterial zone in the same position

Fig. 6―RP-HPLC of fraction C pooled from cation-exchange chromatography on a Hypersil BDS C18 column [The solid line represents absorbance of fractions at 280 nm and the broken line represents a linear gradient of acetonitrile in constant 1% TFA]

Fig.7―RP-HPLC of fraction from C18 RP-HPLC on a Kromasil KR-60 5CN column [The solid line represents fractions absorbance at 280 nm and the broken line represents a linear gradient of acetonitrile in constant 1% TFA]

Fig. 8―MALDI-TOF mass spectra of scolopendrin I

WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE

Fig. 9―Tricine-SDS-PAGE of scolopendrin I [Lane M, molecular mass markers; lane A, scolopendrin I; and lane B, zone of growth inhibition, corresponding to the position of peptide band. The gel was overlaid with viable cells of E.coli K12D31, imbedded in LB agar. Incubation was at 37°C for 24 hr]

(Fig. 9B). The above results suggest that scolopendrin I is a homogeneous antibacterial peptide. The solubility of crude venom in ultrapure water heattreatment at 100°C for 30 min, suggest water solubility and high thermal stability of scolopendrin I. Also, purification of this fraction by cation-exchange chromatography suggests its alkaline nature. However, other properties, such as antimicrobial spectrum, amino acid composition, primary structure, etc. are required be studied further. Scolopendrin I did not show hemolytic activity at the concentration below 30 µM (Fig. 10). The negative control also did not show hemolytic activity. In contrast, the positive control melittin showed 100% hemolysis at a concentration of about 5 µM. On the other hand, hadrurin, an antibacterial peptide from the scorpion showed 75% hemolysis activity at a concentration around 10 µM12. Another antibacterial peptide tachystatin C, from the horseshoe crab showed hemolysis below 25 µM13. Scolopendrin also showed no agglutination activity against mouse erythrocyte at the concentration below 30 µM on macroscopic observation; in contrast, positive control showed haemagglutination activity. Oligo-3-aminopyridine, a potent antibacterial agent against S. aureus has minimum inhibitory concentration (MIC) of 25 µg/ml14. Although the MIC of scolopendrin I was not tested, the MIC of crude venom of Scolopendra subspinipes against S. aureus

93

Fig. 10―Hemolytic activity of scolopendrin I [The samples (2.5 to 30 µM) were mixed with mouse erythrocytes and incubated for 1 hr at 37°C. Hemolytic activity was observed by monitoring the increase in the absorbance at 546 nm. Closed squares represent for scolopendrin I. Open and closed circles represent negative and positive control, respectively. Values are averaged from repeated experiments]

was found to be 5.6 µg/ml, indicating the presence of some more potent antibacterial agents in the venom. References 1 2 3 4 5 6 7 8

9 10 11 12 13

14

Pempberton, R.W (1999) J Ethnopharmacol 65, 207-216 Gomes A, Datta A, Sarangi B, Kar P K& Lahiri S C (1983) Indian J Exp Biol 4, 203-207 Negri A & Llewellyn L (1998) Toxicon, 2, 283-298 You W K, Sohn Y D, Kim K Y, Park D H, Jang Y & Chung K H (2004) Insect Biochem Mol Biol 3, 239-250 Qu X M, Steiner H, Engstrom A, Bennich H & Boman HG (1982) Eur J Biochem 127, 219-224 Liang P S & Dong L H (1988) Chin J Zool 23, 20-21 Hultmark D, Engstrom A, Bennich H, Kapur R & Boman H G (1982) Eur J Biochem 127, 207-217 Moerman L, Bosteels S, Noppe W, Willems J, Clynen E, Schoofs L, Thevissen K, Tytgat J, Van Eldere J, van der Walt J & Verdonck F (2002) Eur J Biochem 269, 4799-4810 Chung K T & Ourth D D (2000) Eur J Biochem 267, 677-683 Schagger H & Von Jagow G (1987) Anal Biochem 166, 368–379 Wang J G, Xia L X & Liu X H (2003) Biotechnol Bull 2, 26-28 Alfredo T L, Gurrola G B, Zamudio F Z & Possani L D (2000) Eur J Biochem 267, 5023-5031 Osaki T, Omotezako M, Nagayama R, Hirata M, Iwanaga S, Kasahara J, Hattori J, Ito I, Sugiyama H & Kawabata S (1999) J Biol Chem, 274, 26172-26178 Cahit A & Ismet K (2004) Indian J Biochem Biophys 41, 120-122