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Isolation and Characterization of a New Member of the Insect Defensin Family from a Beetle, Allomyrina dichotoma
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Isolation and Characterization of a New Member of the Insect Defensin Family from a Beetle, Allomyrina dichotoma Akihiro Miyanoshita, Seiichi Hara,* Masao Sugiyama,1 Ai Asaoka, Kiyoko Taniai, Fumiko Yukuhiro, and Minoru Yamakawa2 Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305, Japan; and *Noda Institute for Scientific Research, Noda, Chiba 278, Japan Received February 14, 1996 A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gramnegative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillinresistant S. aureus (MRSA) isolated from a patient. © 1996 Academic Press, Inc.
Insect defensin is widely distributed in insects of the orders, Coleoptera (1,2), Hymenoptera (3), Hemiptera (4), Diptera (5–10) and Odonata (11). The antibacterial peptide contains cystein residues and forms disulfide bonds. Although insect defensins were originally thought to have structural similarity to mammalian defensins, three-dimensional structures (12) and disulfide bond topologies of the peptides were recently found to be entirely different (9,12). Instead, insect defensins are indicated to be homologous to potassium channel-blocking scorpion toxins such as charybdotoxin (7,13,14). Nuclear magnetic resonance analysis of sapecin, a defensin type antibacterial peptide from the flesh fly, Sarcophaga peregrina, showed that this peptide has a loop, an a-helix and two b-sheets (12). Sapecin is known to interact with bacterial membranes and has a strong affinity for the phospholipid, cardiolipin, suggesting that this peptide causes damage to the bacterial membrane and kills the bacteria (15). Previously, we have isolated and characterized antibacterial peptides from the silkworm, Bombyx mori (16–18). In the course of our search for novel antibacterial peptides from different insect species, we tried to isolate a peptide(s) from the larvae of a beetle, A. dichotoma (Coleoptera). Here, we report the occurrence, structure and biological activity of a new member of the insect defensin family isolated from the hemolymph of A. dichotoma larvae. MATERIALS AND METHODS Biological materials. A. dichotoma third instar larvae were purchased from a local insect shop. As Gram-positive bacteria, Staphylococcus aureus ATCC 6538 P (kindly provided by Dr. M. Yamamoto, Shikibo), Bacillus subtilis ISW 1214 (purchased from Iwaki) and MRSA (kindly provided by T. Sawahata, Tsukuba University Hospital) were cultured at 37°C on a plate containing beef extract 0.5 g (Difco), Peptone 1.0 g (Difco), agar 1.5 g and NaCl 0.5 g (per 100 ml). As Gram-negative bacteria, Escherichia coli JM109 (purchased from Takara) was grown in LB medium and Pseudomonas aeruginosa (kindly provided by Prof. M. Noda, Chiba University School of Medicine) was cultured in the medium described above. Immunization and collection of the hemolymph. A. dichotoma larvae were injected with live E. coli JM 109 (5 × 105
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Present address: Hokko Chemical Industry Central Research Laboratories, Atsugi, Kanagawa 243, Japan. Corresponding author: Fax: +81-298-38-6028; E-mail: [email protected]. 526
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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cells/larva) and reared at 25°C overnight. The hemolymph was collected in ice-cooled 50 ml tubes containing aprotinin (Sigma, 20 mg/ml). After centrifugation at 39000 × g for 50 min to remove the hemocytes, the supernatant was used for the purification of antibacterial peptide(s). Assay for antibacterial activity. Throughout the purification steps, antibacterial activity was analyzed by measuring bacterial growth inhibition zone (19) on thin agarose plates containing beef extract (Difco) with S. aureus ATCC 6538P as an indicator. The ultrasensitive assay (20) was employed to examine the antibacterial spectrum of a purified peptide. MRSA was first cultured at 37°C overnight in 3% tryptic soy broth (TSB, Difco) to observe the effects of a purified peptide on the growth inhibition of MRSA. Then, MRSA (3.5 × 105 cells) was incubated at 37°C with the indicated concentration of the purified peptide in Mueller Hinton broth (Difco, total 100 ml). Ten h after incubation an aliquot (5 ml) of the culture was removed, diluted 10 times with 10 mM phosphate buffer (pH 7.4) containing 130 mM NaCl (PBS) and the absorbance was measured at 550 nm with a Beckman spectrophotometer (DU 650). Twenty h after additional incubation of the bacterial culture, the growth rate was also measured in the same way. Purification of antibacterial peptide. The hemolymph treated at 95°C for 5 min was centrifuged at 16000 × g for 30 min and the supernatant was subjected to the first purification by Sep-Pak C18 cartridge (Waters Associates) pre-equilibrated with 0.05% trifluoroacetic acid (TFA). Proteins were eluted stepwise with 10, 20, 30, 40, 50, 60 and 100% acetonitrile and the antibacterial activity against S. aureus of each fraction was examined. An aliquot (200 ml) containing antibacterial activity was applied to gel-filtration through a column, Sephadex 75 HR 10/30 (FPLC system, Pharmacia) and proteins were eluted with PBS for 120 min. The flow rate was 0.5 ml/min. The antibacterial peak fractions were pooled, loaded onto PepRPC HR 5/5 column (Pharmacia) and eluted for 120 min with a linear gradient of acetonitrile (0–40%) containing 0.05% TFA. The flow rate was 0.25 ml/min. Finally, the antibacterial peak fraction was purified by SMART system using a mRPC C2/C18 PC 3.2/3 column (Pharmacia). The proteins were eluted for 5 min with a linear gradient of acetonitrile (0–20%) containing 0.05% TFA and for 60 min with the same solution (20–30%). The flow rate was 0.1 ml/min. Tricine-SDS-polyacrylamide gel electrophoresis. The purity of an antibacterial peptide was analyzed by 16.5% tricineSDS-polyacrylamide gel electrophoresis according to Schägger and von Jagow (21). The peptide was detected by silver staining using a Silver stain II kit (Wako). Molecular weight markers MW range 2512-16949 (Pharmacia) were used as size markers. Mass spectrometry. The molecular mass of a purified peptide was measured by ion spray mass spectrometry on a Sciex model API-III triple quardrupole mass spectrometer. Amino acid sequencing. Twenty mg of the purified peptide were dissolved in 100 ml of 0.5 M Tris-HCl, pH 8.5, containing 10 mM EDTA and 4 M guanidine hydrochloride, and 1 M dithiothreitol was added. The mixture was flushed with nitrogen gas and incubated for 2 h at room temperature. The pyridylethylated peptide was separated by reverse phase HPLC with a Capcell Pak C8 SG300 column (4.6 × 250 mm, Shiseido) and one-third of the sample remaining was digested with 1 mg of S. sureus V8 protease (Wako) in 100 ml of 50 mM ammonium acetate, pH 4.0, containing 2 mM EDTA for 20 h at 37°C, and the resultant 3 fragments were separated by reverse phase HPLC using the same column. Amino acid sequences were determined by a protein sequencer (Applied Biosystems 473A).
RESULTS We compared the antibacterial activity of the hemolymph from the larvae immunized with S. aureus or with E. coli before purification of the antibacterial peptide(s). The results showed that immunization with E. coli gave higher activity (data not shown). An antibacterial peptide was purified to homogeneity from the hemolymph sample immunized with E. coli, monitoring antibacterial activity against S. aureus. As shown in Fig. 1, the final purification chart indicated a single peak with activity against the bacterium. The peak substance revealed a single stained band in tricine-SDS-polyacrylamide gel electrophoresis for low molecular weight proteins (21), suggesting the presence of a peptide (data not shown). No other protein peaks in the final purification step showed antibacterial activity. About 1 mg of the pure antibacterial peptide was obtained from 2 ml of hemolymph. Amino acid sequence of the purified peptide was determined by a protein sequencer (see Fig. 4). The results showed that this peptide is composed of 43 amino acid residues and contains 6 cystein residues, suggesting that this peptide is a novel member of the insect defensin family (Fig. 4). The molecular mass was also analyzed by ion spray mass spectrometry. The value obtained was 4542.1 ± 1.38 Da and the calculated value from the amino acid sequence was 4544.2 Da, assuming that the C-terminus of the peptide is unmodified. The peptide was isolated from 6 ml of heat-treated hemolymph of larvae injected or not injected with E. coli to examine whether the novel defensin is inducible upon bacterial infection. After 527
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FIG. 1. Final purification of a novel antibacterial peptide by reverse-phase column chromatography. Experimental details were as described under Materials and Methods. Antibacterial activity is shown by the filled column.
reverse phase column chromatography with PepRPC HR 5/5 column (just before the final purification step), the antibacterial fraction from the immunized sample was analyzed by tricine-SDSpolyacryl-amide gel electrophoresis. Although we could not detect antibacterial activity in the sample from non-immunized control hemolymph, the same fraction was analyzed similarly. The results demonstrated that the antibacterial fraction from the immunized sample gave a single stained band corresponding to the novel defensin (Fig. 2, lane B) but no such peptide band was detectable in the same fraction from the non-immunized sample (Fig. 2, lane A). The results suggest that A. dichotoma defensin is inducible upon bacterial infection. The antibacterial spectrum of the purified defensin was examined by the ultrasensitive bacterial
FIG. 2. Effects of immunization on the induction of a novel antibacterial peptide. Protein samples were analyzed by tricine-SDS–polyacrylamide gel electrophoresis. (A) Nonimmunized sample. (B) Immunized sample. The peptide was visualized by silver staining. 528
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growth inhibition assay (20) using S. aureus ATCC 6538P, MRSA, B. subtilis ISW 1214, E. coli JM109 and P. aeruginosa as indicators. The defensin showed antibacterial activity against Grampositive bacteria such as S. aureus and B. subtilis but did not show activity against Gram-negative bacteria such as E. coli and P. aeruginosa (Table 1), indicating a typical antibacterial spectrum of the insect defensin family (9). As our results showed that A. dichotoma defensin can kill MRSA isolated from a patient, we tried to determine the minimal inhibitory concentration (MIC). For this, 10 h after incubation of the indicated amount of defensin with MRSA, an aliquot of the culture (5 ml) was removed and the absorbance was measured at 550 nm. In addition, the absorbance of the culture was also measured 20 h after incubation of the defensin with MRSA. The results revealed that the MIC of A. dichotoma defensin against MRSA was 20 mg/ml, when examined 10 h after incubation of the peptide (Fig. 3). However, the MIC was higher when examined 20 h after incubation (Fig. 3). DISCUSSION The results of amino acid sequencing demonstrate that an antibacterial peptide isolated from the hemolymph of A. dichotoma belongs to the insect defensin family. A. dichotoma defensin exhibits typical antibacterial spectrum of the family. Namely, it acts against Gram-positive bacteria but not against Gram-negative bacteria. The inducibility of A. dichotoma defensin by bacteria is also a typical characteristic of insect antibacterial peptides including insect defensins. One of the interesting features of the antibacterial spectrum of A. dichotoma defensin is that this peptide showed antibacterial activity against MRSA which presently causes a serious infection problem in the medical field of Japan. Although there is no direct evidence yet, we assume that A. dichotoma defensin may interact with cytoplasmic membrane of MRSA like other defensins do (15). Our results of the survey on MIC of defensin against MRSA showed two different values depending upon the incubation time length. The MIC measured after long incubation (20 h) was higher than that measured after shorter incubation (10 h). MRSA is known to contain a higher concentration of proteinases (22) and therefore, the antibacterial peptide may be gradually degraded by the enzymes with incubation time. This might be the reason why we obtained different MICs. Recently, it was reported that MIC against MRSA of a synthetic peptide, KLKLLLLLKLK-NH2 that is a modified a-helix fragment deduced from sapecin B, is about 30 mg/ml (22). This value is higher than that of A. dichotoma defensin. However, the value of natural sapecin C seems to be lower than that of A. dichotoma defensin (50% inhibition concentration of bacterial growth relative to the control is between 0.9–5.4 mg/ml)(7). We compared the amino acid sequences of the various insect defensins isolated from 4 different insect orders (Fig. 4). High identity of insect defensin amino acid sequences suggests that they have TABLE 1 Antibacterial Activity of Allomyrina dichotoma Defensin Bacteria
Gram+/−
Amount (ng/well)
Inhibition-zone diameter (mm)
Staphylococcus aureus ATCC 6538P Staphylococcus aureusa
+
Bacillus subtilis ISW 1214 Escherichia coli JM 109 Pseudomonas aeruginosab
+ −
100 10 100 10 100 10 100
16.33 ± 1.22 4.17 ± 1.59 7.67 ± 0.50 0 9.83 ± 0.42 0 0
−
100
0
+
a Methicillin-resistant Staphylococcus aureus and b Pseudomonas aeruginosa were isolated from patients. Data are the means ±SD of triplicate experiments.
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FIG. 3. Effect of the novel peptide on growth of MRSA. The growth rates of MRSA were measured 10 h (open circle) and 20 h (closed circle) after incubation at 37°C in the presence of various concentrations of the peptide. Each value was normalized against values of the control samples incubated without the peptide (100%). Data are the means of triplicate experiments.
arisen from a common ancestral gene through gene duplications. The identity in amino acid sequence of A. dichotoma defensin with defensins from other Coleoptera is higher than with those from other orders. The results suggest that the score of identity in amino acid sequences between A. dichotoma defensin and other insect defensins exhibited good correlations to the order of
FIG. 4. Comparison of amino acid sequences of the insect defensin family. Gaps are introduced to obtain maximal sequence alignment. Identical amino acid residues in one letter with those of A. dichotoma defensin are boxed. Closed circles indicate the conserved amino acid throughout all defensins. Sequence data of defensins were cited from the following references: 1Moon et al. (1994), 2Bulet et al. (1991), 3Fujiwara et al. (1990), 4Cociancich et al. (1994), 5Lambert et al. (1989), 6Matsuyama and Natori (1988), 7Yamada and Natori (1993), 8Dimarcq et al. (1994), 9Hoffman and Hetru (1992), and 10Lowenberger et al. (1995). A.d., Allomyrina dichotoma; Z. a., Zophobas atratus; P. a., Pyrrhocoris apterus; D. m., Drosophila melanogaster; E. t., Eristalis tenax; and A. a., Aedes aegypti. 530
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phylogenic evolution. Interestingly, all 6 cystein residues and a tyrosine residue next to the Nterminus are completely conserved in 16 insect defensin species. These results suggest that these common amino acid residues might have an important role in the function of insect defensins. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries, Japan (BMP96-V-1-5-2).
REFERENCES 1. Moon, H. J., Lee, S. Y., Kurata, S., Natori, S., and Lee, B. K. (1994) J. Biochem. 116, 53–58. 2. Bulet, P., Cociancich, S., Dimarcq, J.-L., Lambert, J., Reichhart, J.-M., Hoffman, D., Hetru, C., and Hoffman, J. A. (1991) J. Biol. Chem. 266, 24520–24525. 3. Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., and Kobayashi, K. (1990) J. Biol. Chem. 265, 11333–11337. 4. Cociancich, S., Dupont, A., Hegy, G., Lanot, R., Holder, F., Hetru, C., Hoffman, J. A., and Bulet, P. (1994) Biochem. J. 300, 567–575. 5. Lambert, J., Keppi, E., Dimarcq, J.-L., Wicker, C., Reichhart, J.-M., Dunbar, B., Lepage, P., Van Dorsselaer, A., Hoffman, J., Fothergill, J., and Hoffman, D. (1989) Proc. Natl. Acad. Sci. USA 86, 262–266. 6. Matsuyama, K., and Natori, S. (1988) J. Biol. Chem. 263, 17112–17116. 7. Yamada, K., and Natori, S. (1993) Biochem. J. 291, 275–279. 8. Dimarcq, J.-L., Hoffman, D., Meister, M., Bulet, P., Lanot, R., Reichhart, J.-M., and Hoffman, J. A. (1994) Eur. J. Biochem. 221, 201–209. 9. Hoffman, J. A., and Hetru, C. (1992) Immunology Today 13, 201–209. 10. Lowenberger, C., Bulet, P., Charlet, M., Hetru, C., Hodgeman, B., Christensen, B. M., and Hoffman, J. A. (1995) Insect Biochem. Mol. Biol. 25, 867–873. 11. Bulet, P., Cociancich, S., Reuland, M., Sauber, F., Bichoff, R., Hegy, G., Van Dorsselaer, A., Hetru, C., and Hoffman, J. A. (1992) Eur. J. Biochem. 209, 977–984. 12. Hanazawa, H., Shimada, I., Kuzuhara, T., Komano, H., Kohda, D., Inagaki, F., Natori, S., and Arata, Y. (1990) FEBS Lett. 269, 413–420. 13. Bontems, F., Roumestand, C., Gilquin, B., Ménez, A., and Toma, F. (1991) Science 254, 1521–1523. 14. Bontems, F., Roumestand, C., Boyot, P., Gilquin, B., Doljansky, Y., Ménez, A., and Toma, F. (1991) Eur. J. Biochem. 169, 19–28. 15. Matsuyama, K., and Natori, S. (1990) J. Biochem. (Tokyo) 107, 514–518. 16. Hara, S., Taniai, K., Kato, Y., and Yamakawa, M. (1994) Comp. Biochem. Physiol. 108B, 303–308. 17. Hara, S., and Yamakawa, M. (1995) Biochem. J. 310, 651–656. 18. Hara, S., and Yamakawa, M. (1995) J. Biol. Chem. 270, 29923–29927. 19. Hultmark, D., Engström, Å., Bennich, H., Kapur, R., and Boman, H. G. (1982) Eur. J. Biochem. 127, 207–217. 20. Lehrer, R. L., Rosenman, M., Harwig, S. S. S. L., Jackson, R., and Eisenhquer, P. (1991) J. Immunol. Methods 137, 167–173. 21. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368–379. 22. Alvarez-Bravo, J., Kurata, S., and Natori, S. (1994) Biochem. J. 302, 535–538.
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Isolation and Characterization of a New Member of the Insect Defensin Family from a Beetle, Allomyrina dichotoma Akihiro Miyanoshita, Seiichi Hara,* Masao Sugiyama,1 Ai Asaoka, Kiyoko Taniai, Fumiko Yukuhiro, and Minoru Yamakawa2 Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305, Japan; and *Noda Institute for Scientific Research, Noda, Chiba 278, Japan Received February 14, 1996 A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gramnegative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillinresistant S. aureus (MRSA) isolated from a patient. © 1996 Academic Press, Inc.
Insect defensin is widely distributed in insects of the orders, Coleoptera (1,2), Hymenoptera (3), Hemiptera (4), Diptera (5–10) and Odonata (11). The antibacterial peptide contains cystein residues and forms disulfide bonds. Although insect defensins were originally thought to have structural similarity to mammalian defensins, three-dimensional structures (12) and disulfide bond topologies of the peptides were recently found to be entirely different (9,12). Instead, insect defensins are indicated to be homologous to potassium channel-blocking scorpion toxins such as charybdotoxin (7,13,14). Nuclear magnetic resonance analysis of sapecin, a defensin type antibacterial peptide from the flesh fly, Sarcophaga peregrina, showed that this peptide has a loop, an a-helix and two b-sheets (12). Sapecin is known to interact with bacterial membranes and has a strong affinity for the phospholipid, cardiolipin, suggesting that this peptide causes damage to the bacterial membrane and kills the bacteria (15). Previously, we have isolated and characterized antibacterial peptides from the silkworm, Bombyx mori (16–18). In the course of our search for novel antibacterial peptides from different insect species, we tried to isolate a peptide(s) from the larvae of a beetle, A. dichotoma (Coleoptera). Here, we report the occurrence, structure and biological activity of a new member of the insect defensin family isolated from the hemolymph of A. dichotoma larvae. MATERIALS AND METHODS Biological materials. A. dichotoma third instar larvae were purchased from a local insect shop. As Gram-positive bacteria, Staphylococcus aureus ATCC 6538 P (kindly provided by Dr. M. Yamamoto, Shikibo), Bacillus subtilis ISW 1214 (purchased from Iwaki) and MRSA (kindly provided by T. Sawahata, Tsukuba University Hospital) were cultured at 37°C on a plate containing beef extract 0.5 g (Difco), Peptone 1.0 g (Difco), agar 1.5 g and NaCl 0.5 g (per 100 ml). As Gram-negative bacteria, Escherichia coli JM109 (purchased from Takara) was grown in LB medium and Pseudomonas aeruginosa (kindly provided by Prof. M. Noda, Chiba University School of Medicine) was cultured in the medium described above. Immunization and collection of the hemolymph. A. dichotoma larvae were injected with live E. coli JM 109 (5 × 105
1 2
Present address: Hokko Chemical Industry Central Research Laboratories, Atsugi, Kanagawa 243, Japan. Corresponding author: Fax: +81-298-38-6028; E-mail: [email protected]. 526
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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cells/larva) and reared at 25°C overnight. The hemolymph was collected in ice-cooled 50 ml tubes containing aprotinin (Sigma, 20 mg/ml). After centrifugation at 39000 × g for 50 min to remove the hemocytes, the supernatant was used for the purification of antibacterial peptide(s). Assay for antibacterial activity. Throughout the purification steps, antibacterial activity was analyzed by measuring bacterial growth inhibition zone (19) on thin agarose plates containing beef extract (Difco) with S. aureus ATCC 6538P as an indicator. The ultrasensitive assay (20) was employed to examine the antibacterial spectrum of a purified peptide. MRSA was first cultured at 37°C overnight in 3% tryptic soy broth (TSB, Difco) to observe the effects of a purified peptide on the growth inhibition of MRSA. Then, MRSA (3.5 × 105 cells) was incubated at 37°C with the indicated concentration of the purified peptide in Mueller Hinton broth (Difco, total 100 ml). Ten h after incubation an aliquot (5 ml) of the culture was removed, diluted 10 times with 10 mM phosphate buffer (pH 7.4) containing 130 mM NaCl (PBS) and the absorbance was measured at 550 nm with a Beckman spectrophotometer (DU 650). Twenty h after additional incubation of the bacterial culture, the growth rate was also measured in the same way. Purification of antibacterial peptide. The hemolymph treated at 95°C for 5 min was centrifuged at 16000 × g for 30 min and the supernatant was subjected to the first purification by Sep-Pak C18 cartridge (Waters Associates) pre-equilibrated with 0.05% trifluoroacetic acid (TFA). Proteins were eluted stepwise with 10, 20, 30, 40, 50, 60 and 100% acetonitrile and the antibacterial activity against S. aureus of each fraction was examined. An aliquot (200 ml) containing antibacterial activity was applied to gel-filtration through a column, Sephadex 75 HR 10/30 (FPLC system, Pharmacia) and proteins were eluted with PBS for 120 min. The flow rate was 0.5 ml/min. The antibacterial peak fractions were pooled, loaded onto PepRPC HR 5/5 column (Pharmacia) and eluted for 120 min with a linear gradient of acetonitrile (0–40%) containing 0.05% TFA. The flow rate was 0.25 ml/min. Finally, the antibacterial peak fraction was purified by SMART system using a mRPC C2/C18 PC 3.2/3 column (Pharmacia). The proteins were eluted for 5 min with a linear gradient of acetonitrile (0–20%) containing 0.05% TFA and for 60 min with the same solution (20–30%). The flow rate was 0.1 ml/min. Tricine-SDS-polyacrylamide gel electrophoresis. The purity of an antibacterial peptide was analyzed by 16.5% tricineSDS-polyacrylamide gel electrophoresis according to Schägger and von Jagow (21). The peptide was detected by silver staining using a Silver stain II kit (Wako). Molecular weight markers MW range 2512-16949 (Pharmacia) were used as size markers. Mass spectrometry. The molecular mass of a purified peptide was measured by ion spray mass spectrometry on a Sciex model API-III triple quardrupole mass spectrometer. Amino acid sequencing. Twenty mg of the purified peptide were dissolved in 100 ml of 0.5 M Tris-HCl, pH 8.5, containing 10 mM EDTA and 4 M guanidine hydrochloride, and 1 M dithiothreitol was added. The mixture was flushed with nitrogen gas and incubated for 2 h at room temperature. The pyridylethylated peptide was separated by reverse phase HPLC with a Capcell Pak C8 SG300 column (4.6 × 250 mm, Shiseido) and one-third of the sample remaining was digested with 1 mg of S. sureus V8 protease (Wako) in 100 ml of 50 mM ammonium acetate, pH 4.0, containing 2 mM EDTA for 20 h at 37°C, and the resultant 3 fragments were separated by reverse phase HPLC using the same column. Amino acid sequences were determined by a protein sequencer (Applied Biosystems 473A).
RESULTS We compared the antibacterial activity of the hemolymph from the larvae immunized with S. aureus or with E. coli before purification of the antibacterial peptide(s). The results showed that immunization with E. coli gave higher activity (data not shown). An antibacterial peptide was purified to homogeneity from the hemolymph sample immunized with E. coli, monitoring antibacterial activity against S. aureus. As shown in Fig. 1, the final purification chart indicated a single peak with activity against the bacterium. The peak substance revealed a single stained band in tricine-SDS-polyacrylamide gel electrophoresis for low molecular weight proteins (21), suggesting the presence of a peptide (data not shown). No other protein peaks in the final purification step showed antibacterial activity. About 1 mg of the pure antibacterial peptide was obtained from 2 ml of hemolymph. Amino acid sequence of the purified peptide was determined by a protein sequencer (see Fig. 4). The results showed that this peptide is composed of 43 amino acid residues and contains 6 cystein residues, suggesting that this peptide is a novel member of the insect defensin family (Fig. 4). The molecular mass was also analyzed by ion spray mass spectrometry. The value obtained was 4542.1 ± 1.38 Da and the calculated value from the amino acid sequence was 4544.2 Da, assuming that the C-terminus of the peptide is unmodified. The peptide was isolated from 6 ml of heat-treated hemolymph of larvae injected or not injected with E. coli to examine whether the novel defensin is inducible upon bacterial infection. After 527
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FIG. 1. Final purification of a novel antibacterial peptide by reverse-phase column chromatography. Experimental details were as described under Materials and Methods. Antibacterial activity is shown by the filled column.
reverse phase column chromatography with PepRPC HR 5/5 column (just before the final purification step), the antibacterial fraction from the immunized sample was analyzed by tricine-SDSpolyacryl-amide gel electrophoresis. Although we could not detect antibacterial activity in the sample from non-immunized control hemolymph, the same fraction was analyzed similarly. The results demonstrated that the antibacterial fraction from the immunized sample gave a single stained band corresponding to the novel defensin (Fig. 2, lane B) but no such peptide band was detectable in the same fraction from the non-immunized sample (Fig. 2, lane A). The results suggest that A. dichotoma defensin is inducible upon bacterial infection. The antibacterial spectrum of the purified defensin was examined by the ultrasensitive bacterial
FIG. 2. Effects of immunization on the induction of a novel antibacterial peptide. Protein samples were analyzed by tricine-SDS–polyacrylamide gel electrophoresis. (A) Nonimmunized sample. (B) Immunized sample. The peptide was visualized by silver staining. 528
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growth inhibition assay (20) using S. aureus ATCC 6538P, MRSA, B. subtilis ISW 1214, E. coli JM109 and P. aeruginosa as indicators. The defensin showed antibacterial activity against Grampositive bacteria such as S. aureus and B. subtilis but did not show activity against Gram-negative bacteria such as E. coli and P. aeruginosa (Table 1), indicating a typical antibacterial spectrum of the insect defensin family (9). As our results showed that A. dichotoma defensin can kill MRSA isolated from a patient, we tried to determine the minimal inhibitory concentration (MIC). For this, 10 h after incubation of the indicated amount of defensin with MRSA, an aliquot of the culture (5 ml) was removed and the absorbance was measured at 550 nm. In addition, the absorbance of the culture was also measured 20 h after incubation of the defensin with MRSA. The results revealed that the MIC of A. dichotoma defensin against MRSA was 20 mg/ml, when examined 10 h after incubation of the peptide (Fig. 3). However, the MIC was higher when examined 20 h after incubation (Fig. 3). DISCUSSION The results of amino acid sequencing demonstrate that an antibacterial peptide isolated from the hemolymph of A. dichotoma belongs to the insect defensin family. A. dichotoma defensin exhibits typical antibacterial spectrum of the family. Namely, it acts against Gram-positive bacteria but not against Gram-negative bacteria. The inducibility of A. dichotoma defensin by bacteria is also a typical characteristic of insect antibacterial peptides including insect defensins. One of the interesting features of the antibacterial spectrum of A. dichotoma defensin is that this peptide showed antibacterial activity against MRSA which presently causes a serious infection problem in the medical field of Japan. Although there is no direct evidence yet, we assume that A. dichotoma defensin may interact with cytoplasmic membrane of MRSA like other defensins do (15). Our results of the survey on MIC of defensin against MRSA showed two different values depending upon the incubation time length. The MIC measured after long incubation (20 h) was higher than that measured after shorter incubation (10 h). MRSA is known to contain a higher concentration of proteinases (22) and therefore, the antibacterial peptide may be gradually degraded by the enzymes with incubation time. This might be the reason why we obtained different MICs. Recently, it was reported that MIC against MRSA of a synthetic peptide, KLKLLLLLKLK-NH2 that is a modified a-helix fragment deduced from sapecin B, is about 30 mg/ml (22). This value is higher than that of A. dichotoma defensin. However, the value of natural sapecin C seems to be lower than that of A. dichotoma defensin (50% inhibition concentration of bacterial growth relative to the control is between 0.9–5.4 mg/ml)(7). We compared the amino acid sequences of the various insect defensins isolated from 4 different insect orders (Fig. 4). High identity of insect defensin amino acid sequences suggests that they have TABLE 1 Antibacterial Activity of Allomyrina dichotoma Defensin Bacteria
Gram+/−
Amount (ng/well)
Inhibition-zone diameter (mm)
Staphylococcus aureus ATCC 6538P Staphylococcus aureusa
+
Bacillus subtilis ISW 1214 Escherichia coli JM 109 Pseudomonas aeruginosab
+ −
100 10 100 10 100 10 100
16.33 ± 1.22 4.17 ± 1.59 7.67 ± 0.50 0 9.83 ± 0.42 0 0
−
100
0
+
a Methicillin-resistant Staphylococcus aureus and b Pseudomonas aeruginosa were isolated from patients. Data are the means ±SD of triplicate experiments.
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FIG. 3. Effect of the novel peptide on growth of MRSA. The growth rates of MRSA were measured 10 h (open circle) and 20 h (closed circle) after incubation at 37°C in the presence of various concentrations of the peptide. Each value was normalized against values of the control samples incubated without the peptide (100%). Data are the means of triplicate experiments.
arisen from a common ancestral gene through gene duplications. The identity in amino acid sequence of A. dichotoma defensin with defensins from other Coleoptera is higher than with those from other orders. The results suggest that the score of identity in amino acid sequences between A. dichotoma defensin and other insect defensins exhibited good correlations to the order of
FIG. 4. Comparison of amino acid sequences of the insect defensin family. Gaps are introduced to obtain maximal sequence alignment. Identical amino acid residues in one letter with those of A. dichotoma defensin are boxed. Closed circles indicate the conserved amino acid throughout all defensins. Sequence data of defensins were cited from the following references: 1Moon et al. (1994), 2Bulet et al. (1991), 3Fujiwara et al. (1990), 4Cociancich et al. (1994), 5Lambert et al. (1989), 6Matsuyama and Natori (1988), 7Yamada and Natori (1993), 8Dimarcq et al. (1994), 9Hoffman and Hetru (1992), and 10Lowenberger et al. (1995). A.d., Allomyrina dichotoma; Z. a., Zophobas atratus; P. a., Pyrrhocoris apterus; D. m., Drosophila melanogaster; E. t., Eristalis tenax; and A. a., Aedes aegypti. 530
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phylogenic evolution. Interestingly, all 6 cystein residues and a tyrosine residue next to the Nterminus are completely conserved in 16 insect defensin species. These results suggest that these common amino acid residues might have an important role in the function of insect defensins. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries, Japan (BMP96-V-1-5-2).
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