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Purification and Characterization of Lysozyme from Hemolymph of Heliothis virescens Larvae
JOBNAME: BBRC 220#3 PAGE: 1 SESS: 2 OUTPUT: Sun May 5 11:31:50 1996 /xypage/worksmart/tsp000/69829b/6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 502–508 (1996)
0434
Purification and Characterization of Lysozyme from Hemolymph of Heliothis virescens Larvae Timothy D. Lockey and Donald D. Ourth1 Department of Biology, Division of Molecular Sciences and Microbiology, The University of Memphis, Memphis, Tennessee 38152 Received January 30, 1996 Lysozyme is an important antibacterial protein in the insect defense system. Lysozyme was isolated from hemolymph of Heliothis virescens larvae using gel filtration and ion-exchange chromatography. Heliothis lysozyme had a molecular mass of 16,000 daltons by SDS-PAGE. Using acid gel electrophoresis, Heliothis lysozyme migrated faster than egg white lysozyme. The pI of Heliothis lysozyme was estimated as greater than 9.5. Heliothis lysozyme had specific bactericidal activity against three Gram-positive bacteria but no activity against Escherichia coli. The bactericidal activity was stable at 100°C at pH 3.0 after 60 min incubation, but was labile at 100°C at pH 6.8 after 60 min incubation. Heliothis lysozyme was an inducible protein that increased 9 times when comparing unvaccinated with vaccinated larvae. Lysozyme from H. virescens was more similar in molecular mass, heat sensitivity and pH sensitivity to lysozyme isolated from Galleria mellonella and Bombyx mori than to lysozyme isolated from Hyalophora cecropia. © 1996 Academic Press, Inc.
Many different proteins are involved in the immune response of insects (1). The major proteins include the cecropins, attacins, lysozyme and hemolin which are produced in response to bacterial infection or vaccination (2). Lysozyme was first recognized in an insect by Mohrig and Messner (3). Lysozyme was the first immune protein to be isolated from two insect species (4). Although lysozyme has been found in a number of insect species, it has only been purified from a few insects (5). Lysozyme has been purified from Galleria mellonella, Bombyx mori, Hyalophora cecropia, and Manduca sexta (4,6,7). The gene for lysozyme has been isolated and sequenced from H. cecropia (8). The molecular mass of lysozyme from H. cecropia, 13.8 kDa, is similar to egg white lysozyme (5). The increase of lysozyme in hemolymph of vaccinated or infected larvae and the role of lysozyme in the midgut of insects have been demonstrated by a variety of investigators (9–12). In the cotton boll weevil, Anthonomus grandis, lysozyme activity increased after injection of the Gram-negative bacterium Serratia marcescens (13). Lysozyme was also present in eggs of the cotton boll weevil (14). Lysozyme from cecropia pupae was only active against Gram-positive bacteria including Micrococcus lysodeikticus and Bacillus megaterium (15). The primary action of insect lysozyme against Gram-negative bacteria is the hydrolysis of the peptidoglycan cell wall after the bacterium has already been killed by cecropins and attacins (5). Heliothis virescens is an important agricultural pest in the U.S.A. that is highly resistant to nearly all chemical insecticides. Although lysozyme has been characterized in several different lepidopterans, there are no studies describing lysozyme in H. virescens (tobacco budworm) larvae. In this study, we describe the purification and characterization of lysozyme from lepidopteran H. virescens larvae. MATERIALS AND METHODS Maintenance of larvae. Tobacco budworm eggs were obtained from the Bioenvironmental Insect Control Laboratory, U.S. Department of Agriculture, Stoneville, MS. Heliothis virescens larvae were reared on artificial diet (16,17). The larvae were maintained at a constant temperature of 25°C in a 12:12 hr light-dark cycle. After 14 days (5th instar), the larvae were 1
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vaccinated with 1–2 ml containing 10 cells of Enterobacter cloacae. After 24 hr, the larvae where bled by cutting a proleg and collecting the hemolymph into capillary tubes under ice-chilling conditions. The hemolymph was then placed in an Eppendorf tube containing a few crystals of phenylthiourea (PTU) at 4°C. The pooled hemolymph samples were centrifuged at 10,000 rpm for 10 min at 4°C to remove cellular material. The hemolymph supernatant fraction was stored at −80°C. Bacteria. Bacillus megaterium ATCC 14581, Bacillus subtilis ATCC 23059, Bacillus cereus, ATCC 14579 and Micrococcus lysodeikticus were obtained from the American Type Culture Collection (ATCC), Rockville, MD. Escherichia coli K12 D31 was obtained from Dr. Hans Boman, University of Stockholm, Sweden. Lysozyme purification. Gel filtration was done using a 1.5 × 30 cm column packed with Sephadex G-75 (Sigma, St. Louis, MO). Pooled hemolymph from 30 vaccinated larvae (1.5 ml) was applied to the column that had been equilibrated with 0.25 M ammonium acetate, pH 6.8 containing 0.01% PTU. The flow rate was 14 ml/hr. Two ml fractions were collected. The collected fractions were tested for lysozyme activity and at 280 nm for protein absorbance. The column was calibrated with Bio-Rad (Richmond, CA) gel filtration molecular weight standards. The pooled fractions containing lysozyme activity from the gel filtration column were applied to a cation-exchange CM-Sephadex (1.5 × 8 cm) column equilibrated with 0.25 M ammonium acetate, pH 6.8. The lysozyme was eluted from the column using a 100 ml linear gradient of 0.25 M to 1.2 M ammonium acetate, pH 6.8 with a flow rate of 1 ml/min One ml fractions were collected. The collected fractions were tested for lysozyme activity and at 280 nm for protein absorbance. Lysozyme assay. Lysozyme activity was determined by a diffusion assay done in agar plates. The well-diffusion assay, done in petri plates, consisted of 10 ml of tryptic soy agar (TSA) (Difco, Detroit, MI) containing 2.5 × 105 cells of M. lysodeikticus. Wells (3 mm in diameter) were cut in the agar and the agar plugs then removed. The wells were filled with 10 ml of the column fractions. After 48 hr. at 30°C, the zone of inhibition was measured in mm using a Kallestad (Chaska, MN) calibrated viewer. A standard curve was prepared by serially diluting a 1 mg/ml stock solution of egg white lysozyme. The lowest detectable level for this method was 0.6 mg/ml of egg white lysozyme. Heliothis lysozyme activity against both Gram-positive and Gram-negative bacteria was determined using the well diffusion assay in which 2.5 × 105 cells of B. megaterium or B. cereus or B. subtilis or E. coli were added to 10 ml of molten TSA. A purified Heliothis lysozyme solution (250 mg/ml) in 0.1 M sodium phosphate, pH 6.8 was prepared. Ten ml of the lysozyme solution were placed in each well. The zones of inhibition were compared to those produced by 10 ml of a 250 mg/ml solution of egg white lysozyme. The following procedure was used to determine the temperature stability of Heliothis lysozyme. A solution of purified Heliothis lysozyme (25 mg/ml) was incubated at temperatures of 25, 45, 60 or 100°C. At specific times (0, 5, 15, 30 and 60 min), a 10 ml sample was removed and assayed for the amount of lysozyme activity present using the agar well diffusion assay. Triplicate assays were done. Acid gel electrophoresis. Acid polyacrylamide gel electrophoresis was carried out as described by Hultmark et al. (1980). The gel was prepared using a b-alanine-acetic acid buffer, pH 4.3 having a gel concentration of 15% (60:0.8 acrylamide:bis ratio). The acid gel (8 × 10 cm) was 1.5 mm thick. Electrophoresis was performed for 2 hr. at 200 volts. Samples were acidified with a 0.5–1.0 volume of 1 N acetic acid. The acid gel was either stained for protein with Coomassie blue or lysozyme was visualized using an activity assay after first washing the gel two times in 0.1 M sodium phosphate buffer, pH 7.2 for 10 min each time. This was followed by overlaying the gel with 10 ml of TSA containing 2.5 × 105 cells of M. lysodeikticus. The lysozyme bands were detected after 48 hrs. by clearing of bacteria in the overlaid agarose gel. Molecular weight determination. Molecular weight of the Heliothis lysozyme was determined using an SDSpolyacrylamide gel electrophoresis method for low molecular weight proteins (modification of the discontinuous procedure of Schägger and Von Jagow, Sigma Technical bulletin MWM-100) (18). The SDS gel had a separating gel of 16.5% acrylamide with a 10% acrylamide spacer gel and a 4% stacking gel, pH 8.45. The gel (8 × 10 cm and 0.75 mm thick) was subjected to electrophoresis for 2 hrs. at 90 mAmps. The gel was stained for protein with Coomassie blue. Low molecular weight standards (Bio-Rad Laboratories, Hercules, CA) were phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg ovalbumin (43 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (22 kDa) and egg white lysozyme (14 kDa). Myoglobin digest standards (Pharmacia, Piscataway, NJ) consisted of myoglobin (17 kDa), myoglobin I & II (14 kDa), myoglobin I (8.1 kDa), myoglobin II (6.2 kDa) and myoglobin III (2.5 kDa). Protein assay. Protein concentration of the purified Heliothis lysozyme was determined using the BCA protein assay (Pierce, Rockford, IL). Bovine serum albumin was used as the standard. Isoelectric focusing. The isoelectric point of Heliothis lysozyme was estimated using a horizontal electrophoresis cell with Bio-Lyte 3/10 ampholytes (Bio-Rad, Laboratories, Hercules, CA). 6
RESULTS The initial step in purification of lysozyme from hemolymph of vaccinated larvae began with gel filtration chromatography. This was done to remove proteins that might later interfere with cationexchange chromatography. When immune hemolymph from H. virescens was applied to a Sephadex G-75 gel filtration column equilibrated with 0.25 M ammonium acetate buffer, pH 6.8, two 503
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main protein peaks were seen (Fig. 1). Lysozyme activity was detected in the trough between the two protein peaks. The tubes containing lysozyme activity corresponded to a molecular mass of approximately 10,000 daltons based on the Bio-Rad gel filtration standards. The tubes containing lysozyme activity were pooled and applied to a cation-exchange column. The CM-Sephadex ion-exchange column was first equilibrated with 0.25 M ammonium acetate buffer, pH 6.8. The pooled fractions from gel filtration were applied to the ion-exchange column and the column then washed with 0.25 M ammonium acetate buffer. The lysozyme was eluted from the column with a linear gradient of 0.25 M to 1.2 M ammonium acetate buffer, pH 6.8. Heliothis lysozyme eluted from the column as a single peak (Fig. 2). The pooled Heliothis lysozyme fractions from the single peak obtained by ion-exchange chromatography were lyophilized and dialyzed in 10 mM sodium phosphate buffer, pH 7.2 to remove ammonium acetate. The purified lysozyme was characterized by acid gel electrophoresis and SDS-PAGE to determine the purity of the lysozyme. Results of acid gel electrophoresis are shown in Figure 3. The purified Heliothis lysozyme produced a single band that migrated slightly faster than egg white lysozyme. A zone of clearing was obtained with the acid gel overlay that corresponded to the lysozyme protein band (Fig. 3). The molecular mass of lysozyme from H. virescens was determined to be 16,000 daltons by SDS-PAGE using the low molecular weight standards (Fig. 4). The isoelectric point of Heliothis lysozyme was estimated to be greater than 9.5 as the protein migrated to the end of the basic region of the gel. Specific activity of Heliothis lysozyme against the Gram-positive bacteria tested showed that Heliothis lysozyme is active against M. lysodeikticus, B. megaterium and B. subtilis. No lysozyme activity was detected against B. cereus and E. coli K12 D31 (Table 1). Lysozyme from Heliothis was very stable at high temperatures and low pH. There were no decrease in lysozyme activity at 25, 45 and 60°C at either pH 3.0 or pH 6.8. At 100°C, lysozyme was stable at pH 3. But at 100°C, pH 6.8, the amount of lysozyme activity decreased after 45 min to undetectable levels at 60 min (Fig. 5). After vaccination of H. virescens larvae with E. cloacae, the concentration of lysozyme in hemolymph increased from 6.6 mg/ml in unvaccinated larvae to 59.9 mg/ml in vaccinated larvae.
FIG. 1. Gel filtration with Sephadex G-75. Immune hemolymph (1.5 ml) was applied to a 1.5 × 30 cm column equilibrated with 0.25 M ammonium acetate, pH 6.8. Flow rate was 14 ml/hr. Two-milliliter fractions were collected and assayed for lysozyme activity and at 280 nm for protein. The tubes (15–20) containing lysozyme were pooled. 504
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FIG. 2. CM-cation exchange chromatography. Pooled fractions from gel filtration (Fig. 1) were applied to a column (1.5 × 8 cm) that was equilibrated with 0.25 M ammonium acetate, pH 6.8. The column was eluted with a gradient of 50 ml of 0.25 M ammonium acetate, pH 6.8 and 50 ml of 1.2 M ammonium acetate, pH 6.8. One milliliter fractions were collected and assayed for lysozyme activity and at 280 nm for protein.
The lysozyme concentrations were calculated from a standard curve for egg white lysozyme using the well diffusion assay. DISCUSSION Lysozyme has been isolated and characterized from several insects species (4,6,7). In this study, we have purified and characterized lysozyme from hemolymph of H. virescens larvae. The purification of lysozyme was done using standard techniques for the purification of immune proteins from insects. Gel filtration using Sephadex G-75 (Fig. 1) removed high molecular weight proteins, including phenoloxidase, which would cause melanization of hemolymph and interfere with column flow. Lysozyme activity from the gel filtration column was further separated by CM-ion
FIG. 3. Acid gel electrophoresis at pH 4.3. (A) Overlay with 10 ml of tryptic soy agar containing 2.5 × 105 cells of Micrococcus lysodeikticus. Arrow indicates zone of clearing (killing) of bacteria in agarose overlay. (B) Stained for protein with Coomassie blue. Lane 1, 225 mg (2.5 ml) of immune hemolymph; Lane 2, 1.9 mg (15 ml) of purified Heliothis lysozyme; Lane 3, 2.5 mg (15 ml) egg white lysozyme. Samples were acidified with 0.5–1.0 volumes of 1 N acetic acid. 505
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FIG. 4. SDS-PAGE gel for low molecular weight proteins. Lane 1, purified Heliothis lysozyme; Lane 2, egg white lysozyme; Lane 3, immune hemolymph. Molecular weight markers are a combination of Bio-Rad low molecular weight markers and Pharmacia myoglobin digest standards.
exchange chromatography and purified into a single peak (Fig. 2). By acid gel electrophoresis, Heliothis lysozyme migrated slightly faster than egg white lysozyme (Fig. 3). The Heliothis lysozyme was more similar in migration on an acid gel to lysozyme from Galleria mellonella (4) and Trichoplusia ni (19) than to lysozyme from H. cecropia (6). Hultmark et al. (1980) found that lysozyme from H. cecropia migrated similarly to egg white lysozyme. The molecular mass of Heliothis lysozyme was determined to be 16,000 daltons (Fig. 4). This is similar to the molecular mass of B. mori lysozyme (16,500 daltons) (4), but greater than the molecular mass of H. cecropia lysozyme (13,800 daltons) (20) and G. mellonella lysozyme (14,700 daltons) (21). The heat stability tests of lysozyme demonstrated that it was stable at 100°C for 60 min at pH 3.0 (Fig. 5). At a higher pH of 6.8, lysozyme was found to be less stable at the higher temperature of 100°C (Fig. 5). The heat stability results obtained here were similar to those obtained for lysozyme from G. mellonella and B. mori (4). Bactericidal activity was found against M. lysodeikticus, B. megaterium and B. subtilis (Table 1). There was no bactericidal activity against the Gram-positive bacterium (B. cereus) or against the Gram-negative bacterium (E. coli K12 D31). Many studies have been done to determine the response of lysozyme in insects to infection or vaccination. Lysozyme is most often found at low levels in normal hemolymph and to be induced in response to injection of bacteria into the hemolymph. Large lesions, seen by electron microscopy, were produced in the cell envelope of E. coli K12 D31 by Heliothis immune hemolymph TABLE 1 Lysozyme Activity against Different Bacteria Zone of inhibition (mm) Bacteria Micrococcus lysodeikticus Bacillus megaterium Bacillus subtilis Bacillus cereus Escherichia coli Saline control
Heliothis lysozyme* a
Egg white lysozyme* a
13.4 ± 1.4 9.7 ± 0.8 4.2 ± 0.2 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
7.7 ± 1.0 9.2 ± 0.1 5.1 ± 0.1 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
Note. Lysozyme activity was determined against Gram-positive and Gramnegative bacteria. The well diffusion assay was used to determine lysozyme activity. Agar plates containing 2.5 × 105 cells in tryptic soy agar were used. Well diameter was 3 mm. Results are the mean of three replicates ±SD. a Zone of clearing at a concentration of 250 mg/ml of lysozyme. 506
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FIG. 5. Temperature stability of Heliothis lysozyme. Purified lysozyme was incubated at different temperatures (60°C and 100°C) and at different times (0, 5, 15, 30 and 60 min.). Aliquots were removed at the different times and assayed for lysozyme activity. Data represent the mean of three replicates.
(22). Heliothis immune hemolymph contains lysozyme and antibacterial peptides (22). Lysozyme is mainly found in the hemolymph but has also been identified in the pericardial cells of M. sexta (7), in the gut of several insects (10) and in eggs from the cotton boll weevil (14). In hemolymph from unvaccinated H. virescens larvae, the lysozyme concentration was 6.6 mg/ml. The hemolymph lysozyme concentration greatly increased to 59.9 mg/ml after the Heliothis larvae were vaccinated with E. cloacae. A 9-fold increase in lysozyme concentration was therefore seen when comparing unvaccinated with vaccinated larvae indicating an inducible protein. Even though lysozyme is found to be ubiquitous in insects, there are nevertheless chemical and physical differences in the lysozymes isolated from different insect species within the Lepidopteran order. Lysozyme from H. virescens was compared to lysozymes from other insects. Heliothis lysozyme is more similar in molecular weight and heat sensitivity to lysozyme isolated from B. mori than to lysozyme found in H. cecropia or G. mellonella. Migration of H. virescens lysozyme on an acid gel was faster than egg white lysozyme and its molecular mass was determined to be 16,000 daltons by SDS-PAGE. Heliothis lysozyme was very resistant to heat denaturation and was stable at an acid pH. The isoelectric point of Heliothis lysozyme was estimated as greater than 9.5. This compares with the pl of H. cecropia lysozyme which was also determined to be greater than 9.5 (6). Inducible proteins like lysozyme, cecropin and attacin are well characterized proteins which have a role in defense against bacterial infections (1,2). Although lysozyme is directly active against a few Gram-positive bacteria, its main function is hydrolysis of the peptidoglycan cell wall after bacteria have already been killed by other insect defensive proteins and phagocytosis (20). Two insect immune proteins have recently been identified in larvae of H. virescens as cecropinlike and attacin-like proteins (22–25). Along with these two antibacterial proteins and the purification of lysozyme in this study, we have thereby documented the existence of the three major inducible immune proteins in H. virescens. By determining the important proteins of this insect immune system, we can better elucidate the roles of the individual proteins in the Heliothis defensive response to bacterial infections. REFERENCES 1. Dunn, P. E. (1990) BioScience 40, 738–744. 2. Boman, H. G., and Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103–126. 507
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Mohrig, W., and Messner, B. (1968) Biol. Zentralbl. 87, 705–718. Powning, R. F., and Davidson, W. J. (1973) Comp. Biochem. Physiol. 45B, 669–686. Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.-Y., and Lidholm, D.-A. (1991) Eur. J. Biochem. 201, 23–31. Hultmark, D., Steiner, H., Rasmuson, T., and Boman, H. G. (1980) Eur. J. Biochem. 106, 7–16. Russell, V. W., and Dunn, P. E. (1990) Eur. J. Biochem. 20, 501–509. Sun, S.-C., Åsling, B., and Faye, I. (1991) J. Biol. Chem. 266, 6644–6649. Kaaya, G. P., Flyg, C., and Boman, H. G. (1987) Insect Biochem. 17, 309–315. Lemos, F. J. A., and Terra, W. R. (1991) Comp. Biochem. Physiol. B 100, 265–268. Pryce, M. J., Aston, W. P., and Chadwick, J. S. (1990) Dev. Comp. Immunol. 14, 369–378. Xylander, W. E. R., and Nevermann, L. (1990) J. Invertebr. Pathol. 56, 206–214. Ourth, D. D., and Smalley, D. L. (1980) J. Invertebr. Pathol. 36, 104–112. Ourth, D. D., and Jones, B. R. (1980) Experientia 36, 196. Boman, H. G., and Hultmark, D. (1981) TIBS 6, 306–309. Ourth, D. D. (1988) J. Econ. Entomol. 81, 148–151. Raulston, J. R., and King, E. G. (1984) Advances and Challenges in Insect Rearing, USDA-ARS, New Orleans, LA. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368–379. Andersons, D., Gunne, H., Hellers, M., Johansson, H., and Steiner, H. (1990) Insect Biochem. 20, 537–543. Boman, H. G. (1991) Cell 65, 205–207. Powning, R. F., and Davidson, W. J. (1976) Comp. Biochem. Physiol. 55B, 221–228. Ourth, D. D., Lockey, T. D., and Renis, H. E. (1994) Biochem. Biophys. Res. Comm. 200, 35–44. Lockey, T. D., and Ourth, D. D. (1993) J. Immunol. 150, 314A. Lockey, T. D., and Ourth, D. D. (1994) Dev. Comp. Immunol. 18, S124A. Lockey, T. D., and Ourth, D. D. (1996) Eur. J. Biochem., in press.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 502–508 (1996)
0434
Purification and Characterization of Lysozyme from Hemolymph of Heliothis virescens Larvae Timothy D. Lockey and Donald D. Ourth1 Department of Biology, Division of Molecular Sciences and Microbiology, The University of Memphis, Memphis, Tennessee 38152 Received January 30, 1996 Lysozyme is an important antibacterial protein in the insect defense system. Lysozyme was isolated from hemolymph of Heliothis virescens larvae using gel filtration and ion-exchange chromatography. Heliothis lysozyme had a molecular mass of 16,000 daltons by SDS-PAGE. Using acid gel electrophoresis, Heliothis lysozyme migrated faster than egg white lysozyme. The pI of Heliothis lysozyme was estimated as greater than 9.5. Heliothis lysozyme had specific bactericidal activity against three Gram-positive bacteria but no activity against Escherichia coli. The bactericidal activity was stable at 100°C at pH 3.0 after 60 min incubation, but was labile at 100°C at pH 6.8 after 60 min incubation. Heliothis lysozyme was an inducible protein that increased 9 times when comparing unvaccinated with vaccinated larvae. Lysozyme from H. virescens was more similar in molecular mass, heat sensitivity and pH sensitivity to lysozyme isolated from Galleria mellonella and Bombyx mori than to lysozyme isolated from Hyalophora cecropia. © 1996 Academic Press, Inc.
Many different proteins are involved in the immune response of insects (1). The major proteins include the cecropins, attacins, lysozyme and hemolin which are produced in response to bacterial infection or vaccination (2). Lysozyme was first recognized in an insect by Mohrig and Messner (3). Lysozyme was the first immune protein to be isolated from two insect species (4). Although lysozyme has been found in a number of insect species, it has only been purified from a few insects (5). Lysozyme has been purified from Galleria mellonella, Bombyx mori, Hyalophora cecropia, and Manduca sexta (4,6,7). The gene for lysozyme has been isolated and sequenced from H. cecropia (8). The molecular mass of lysozyme from H. cecropia, 13.8 kDa, is similar to egg white lysozyme (5). The increase of lysozyme in hemolymph of vaccinated or infected larvae and the role of lysozyme in the midgut of insects have been demonstrated by a variety of investigators (9–12). In the cotton boll weevil, Anthonomus grandis, lysozyme activity increased after injection of the Gram-negative bacterium Serratia marcescens (13). Lysozyme was also present in eggs of the cotton boll weevil (14). Lysozyme from cecropia pupae was only active against Gram-positive bacteria including Micrococcus lysodeikticus and Bacillus megaterium (15). The primary action of insect lysozyme against Gram-negative bacteria is the hydrolysis of the peptidoglycan cell wall after the bacterium has already been killed by cecropins and attacins (5). Heliothis virescens is an important agricultural pest in the U.S.A. that is highly resistant to nearly all chemical insecticides. Although lysozyme has been characterized in several different lepidopterans, there are no studies describing lysozyme in H. virescens (tobacco budworm) larvae. In this study, we describe the purification and characterization of lysozyme from lepidopteran H. virescens larvae. MATERIALS AND METHODS Maintenance of larvae. Tobacco budworm eggs were obtained from the Bioenvironmental Insect Control Laboratory, U.S. Department of Agriculture, Stoneville, MS. Heliothis virescens larvae were reared on artificial diet (16,17). The larvae were maintained at a constant temperature of 25°C in a 12:12 hr light-dark cycle. After 14 days (5th instar), the larvae were 1
To whom correspondence should be addressed. Fax: (901)678-4457. 502
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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vaccinated with 1–2 ml containing 10 cells of Enterobacter cloacae. After 24 hr, the larvae where bled by cutting a proleg and collecting the hemolymph into capillary tubes under ice-chilling conditions. The hemolymph was then placed in an Eppendorf tube containing a few crystals of phenylthiourea (PTU) at 4°C. The pooled hemolymph samples were centrifuged at 10,000 rpm for 10 min at 4°C to remove cellular material. The hemolymph supernatant fraction was stored at −80°C. Bacteria. Bacillus megaterium ATCC 14581, Bacillus subtilis ATCC 23059, Bacillus cereus, ATCC 14579 and Micrococcus lysodeikticus were obtained from the American Type Culture Collection (ATCC), Rockville, MD. Escherichia coli K12 D31 was obtained from Dr. Hans Boman, University of Stockholm, Sweden. Lysozyme purification. Gel filtration was done using a 1.5 × 30 cm column packed with Sephadex G-75 (Sigma, St. Louis, MO). Pooled hemolymph from 30 vaccinated larvae (1.5 ml) was applied to the column that had been equilibrated with 0.25 M ammonium acetate, pH 6.8 containing 0.01% PTU. The flow rate was 14 ml/hr. Two ml fractions were collected. The collected fractions were tested for lysozyme activity and at 280 nm for protein absorbance. The column was calibrated with Bio-Rad (Richmond, CA) gel filtration molecular weight standards. The pooled fractions containing lysozyme activity from the gel filtration column were applied to a cation-exchange CM-Sephadex (1.5 × 8 cm) column equilibrated with 0.25 M ammonium acetate, pH 6.8. The lysozyme was eluted from the column using a 100 ml linear gradient of 0.25 M to 1.2 M ammonium acetate, pH 6.8 with a flow rate of 1 ml/min One ml fractions were collected. The collected fractions were tested for lysozyme activity and at 280 nm for protein absorbance. Lysozyme assay. Lysozyme activity was determined by a diffusion assay done in agar plates. The well-diffusion assay, done in petri plates, consisted of 10 ml of tryptic soy agar (TSA) (Difco, Detroit, MI) containing 2.5 × 105 cells of M. lysodeikticus. Wells (3 mm in diameter) were cut in the agar and the agar plugs then removed. The wells were filled with 10 ml of the column fractions. After 48 hr. at 30°C, the zone of inhibition was measured in mm using a Kallestad (Chaska, MN) calibrated viewer. A standard curve was prepared by serially diluting a 1 mg/ml stock solution of egg white lysozyme. The lowest detectable level for this method was 0.6 mg/ml of egg white lysozyme. Heliothis lysozyme activity against both Gram-positive and Gram-negative bacteria was determined using the well diffusion assay in which 2.5 × 105 cells of B. megaterium or B. cereus or B. subtilis or E. coli were added to 10 ml of molten TSA. A purified Heliothis lysozyme solution (250 mg/ml) in 0.1 M sodium phosphate, pH 6.8 was prepared. Ten ml of the lysozyme solution were placed in each well. The zones of inhibition were compared to those produced by 10 ml of a 250 mg/ml solution of egg white lysozyme. The following procedure was used to determine the temperature stability of Heliothis lysozyme. A solution of purified Heliothis lysozyme (25 mg/ml) was incubated at temperatures of 25, 45, 60 or 100°C. At specific times (0, 5, 15, 30 and 60 min), a 10 ml sample was removed and assayed for the amount of lysozyme activity present using the agar well diffusion assay. Triplicate assays were done. Acid gel electrophoresis. Acid polyacrylamide gel electrophoresis was carried out as described by Hultmark et al. (1980). The gel was prepared using a b-alanine-acetic acid buffer, pH 4.3 having a gel concentration of 15% (60:0.8 acrylamide:bis ratio). The acid gel (8 × 10 cm) was 1.5 mm thick. Electrophoresis was performed for 2 hr. at 200 volts. Samples were acidified with a 0.5–1.0 volume of 1 N acetic acid. The acid gel was either stained for protein with Coomassie blue or lysozyme was visualized using an activity assay after first washing the gel two times in 0.1 M sodium phosphate buffer, pH 7.2 for 10 min each time. This was followed by overlaying the gel with 10 ml of TSA containing 2.5 × 105 cells of M. lysodeikticus. The lysozyme bands were detected after 48 hrs. by clearing of bacteria in the overlaid agarose gel. Molecular weight determination. Molecular weight of the Heliothis lysozyme was determined using an SDSpolyacrylamide gel electrophoresis method for low molecular weight proteins (modification of the discontinuous procedure of Schägger and Von Jagow, Sigma Technical bulletin MWM-100) (18). The SDS gel had a separating gel of 16.5% acrylamide with a 10% acrylamide spacer gel and a 4% stacking gel, pH 8.45. The gel (8 × 10 cm and 0.75 mm thick) was subjected to electrophoresis for 2 hrs. at 90 mAmps. The gel was stained for protein with Coomassie blue. Low molecular weight standards (Bio-Rad Laboratories, Hercules, CA) were phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg ovalbumin (43 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (22 kDa) and egg white lysozyme (14 kDa). Myoglobin digest standards (Pharmacia, Piscataway, NJ) consisted of myoglobin (17 kDa), myoglobin I & II (14 kDa), myoglobin I (8.1 kDa), myoglobin II (6.2 kDa) and myoglobin III (2.5 kDa). Protein assay. Protein concentration of the purified Heliothis lysozyme was determined using the BCA protein assay (Pierce, Rockford, IL). Bovine serum albumin was used as the standard. Isoelectric focusing. The isoelectric point of Heliothis lysozyme was estimated using a horizontal electrophoresis cell with Bio-Lyte 3/10 ampholytes (Bio-Rad, Laboratories, Hercules, CA). 6
RESULTS The initial step in purification of lysozyme from hemolymph of vaccinated larvae began with gel filtration chromatography. This was done to remove proteins that might later interfere with cationexchange chromatography. When immune hemolymph from H. virescens was applied to a Sephadex G-75 gel filtration column equilibrated with 0.25 M ammonium acetate buffer, pH 6.8, two 503
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main protein peaks were seen (Fig. 1). Lysozyme activity was detected in the trough between the two protein peaks. The tubes containing lysozyme activity corresponded to a molecular mass of approximately 10,000 daltons based on the Bio-Rad gel filtration standards. The tubes containing lysozyme activity were pooled and applied to a cation-exchange column. The CM-Sephadex ion-exchange column was first equilibrated with 0.25 M ammonium acetate buffer, pH 6.8. The pooled fractions from gel filtration were applied to the ion-exchange column and the column then washed with 0.25 M ammonium acetate buffer. The lysozyme was eluted from the column with a linear gradient of 0.25 M to 1.2 M ammonium acetate buffer, pH 6.8. Heliothis lysozyme eluted from the column as a single peak (Fig. 2). The pooled Heliothis lysozyme fractions from the single peak obtained by ion-exchange chromatography were lyophilized and dialyzed in 10 mM sodium phosphate buffer, pH 7.2 to remove ammonium acetate. The purified lysozyme was characterized by acid gel electrophoresis and SDS-PAGE to determine the purity of the lysozyme. Results of acid gel electrophoresis are shown in Figure 3. The purified Heliothis lysozyme produced a single band that migrated slightly faster than egg white lysozyme. A zone of clearing was obtained with the acid gel overlay that corresponded to the lysozyme protein band (Fig. 3). The molecular mass of lysozyme from H. virescens was determined to be 16,000 daltons by SDS-PAGE using the low molecular weight standards (Fig. 4). The isoelectric point of Heliothis lysozyme was estimated to be greater than 9.5 as the protein migrated to the end of the basic region of the gel. Specific activity of Heliothis lysozyme against the Gram-positive bacteria tested showed that Heliothis lysozyme is active against M. lysodeikticus, B. megaterium and B. subtilis. No lysozyme activity was detected against B. cereus and E. coli K12 D31 (Table 1). Lysozyme from Heliothis was very stable at high temperatures and low pH. There were no decrease in lysozyme activity at 25, 45 and 60°C at either pH 3.0 or pH 6.8. At 100°C, lysozyme was stable at pH 3. But at 100°C, pH 6.8, the amount of lysozyme activity decreased after 45 min to undetectable levels at 60 min (Fig. 5). After vaccination of H. virescens larvae with E. cloacae, the concentration of lysozyme in hemolymph increased from 6.6 mg/ml in unvaccinated larvae to 59.9 mg/ml in vaccinated larvae.
FIG. 1. Gel filtration with Sephadex G-75. Immune hemolymph (1.5 ml) was applied to a 1.5 × 30 cm column equilibrated with 0.25 M ammonium acetate, pH 6.8. Flow rate was 14 ml/hr. Two-milliliter fractions were collected and assayed for lysozyme activity and at 280 nm for protein. The tubes (15–20) containing lysozyme were pooled. 504
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FIG. 2. CM-cation exchange chromatography. Pooled fractions from gel filtration (Fig. 1) were applied to a column (1.5 × 8 cm) that was equilibrated with 0.25 M ammonium acetate, pH 6.8. The column was eluted with a gradient of 50 ml of 0.25 M ammonium acetate, pH 6.8 and 50 ml of 1.2 M ammonium acetate, pH 6.8. One milliliter fractions were collected and assayed for lysozyme activity and at 280 nm for protein.
The lysozyme concentrations were calculated from a standard curve for egg white lysozyme using the well diffusion assay. DISCUSSION Lysozyme has been isolated and characterized from several insects species (4,6,7). In this study, we have purified and characterized lysozyme from hemolymph of H. virescens larvae. The purification of lysozyme was done using standard techniques for the purification of immune proteins from insects. Gel filtration using Sephadex G-75 (Fig. 1) removed high molecular weight proteins, including phenoloxidase, which would cause melanization of hemolymph and interfere with column flow. Lysozyme activity from the gel filtration column was further separated by CM-ion
FIG. 3. Acid gel electrophoresis at pH 4.3. (A) Overlay with 10 ml of tryptic soy agar containing 2.5 × 105 cells of Micrococcus lysodeikticus. Arrow indicates zone of clearing (killing) of bacteria in agarose overlay. (B) Stained for protein with Coomassie blue. Lane 1, 225 mg (2.5 ml) of immune hemolymph; Lane 2, 1.9 mg (15 ml) of purified Heliothis lysozyme; Lane 3, 2.5 mg (15 ml) egg white lysozyme. Samples were acidified with 0.5–1.0 volumes of 1 N acetic acid. 505
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FIG. 4. SDS-PAGE gel for low molecular weight proteins. Lane 1, purified Heliothis lysozyme; Lane 2, egg white lysozyme; Lane 3, immune hemolymph. Molecular weight markers are a combination of Bio-Rad low molecular weight markers and Pharmacia myoglobin digest standards.
exchange chromatography and purified into a single peak (Fig. 2). By acid gel electrophoresis, Heliothis lysozyme migrated slightly faster than egg white lysozyme (Fig. 3). The Heliothis lysozyme was more similar in migration on an acid gel to lysozyme from Galleria mellonella (4) and Trichoplusia ni (19) than to lysozyme from H. cecropia (6). Hultmark et al. (1980) found that lysozyme from H. cecropia migrated similarly to egg white lysozyme. The molecular mass of Heliothis lysozyme was determined to be 16,000 daltons (Fig. 4). This is similar to the molecular mass of B. mori lysozyme (16,500 daltons) (4), but greater than the molecular mass of H. cecropia lysozyme (13,800 daltons) (20) and G. mellonella lysozyme (14,700 daltons) (21). The heat stability tests of lysozyme demonstrated that it was stable at 100°C for 60 min at pH 3.0 (Fig. 5). At a higher pH of 6.8, lysozyme was found to be less stable at the higher temperature of 100°C (Fig. 5). The heat stability results obtained here were similar to those obtained for lysozyme from G. mellonella and B. mori (4). Bactericidal activity was found against M. lysodeikticus, B. megaterium and B. subtilis (Table 1). There was no bactericidal activity against the Gram-positive bacterium (B. cereus) or against the Gram-negative bacterium (E. coli K12 D31). Many studies have been done to determine the response of lysozyme in insects to infection or vaccination. Lysozyme is most often found at low levels in normal hemolymph and to be induced in response to injection of bacteria into the hemolymph. Large lesions, seen by electron microscopy, were produced in the cell envelope of E. coli K12 D31 by Heliothis immune hemolymph TABLE 1 Lysozyme Activity against Different Bacteria Zone of inhibition (mm) Bacteria Micrococcus lysodeikticus Bacillus megaterium Bacillus subtilis Bacillus cereus Escherichia coli Saline control
Heliothis lysozyme* a
Egg white lysozyme* a
13.4 ± 1.4 9.7 ± 0.8 4.2 ± 0.2 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
7.7 ± 1.0 9.2 ± 0.1 5.1 ± 0.1 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
Note. Lysozyme activity was determined against Gram-positive and Gramnegative bacteria. The well diffusion assay was used to determine lysozyme activity. Agar plates containing 2.5 × 105 cells in tryptic soy agar were used. Well diameter was 3 mm. Results are the mean of three replicates ±SD. a Zone of clearing at a concentration of 250 mg/ml of lysozyme. 506
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FIG. 5. Temperature stability of Heliothis lysozyme. Purified lysozyme was incubated at different temperatures (60°C and 100°C) and at different times (0, 5, 15, 30 and 60 min.). Aliquots were removed at the different times and assayed for lysozyme activity. Data represent the mean of three replicates.
(22). Heliothis immune hemolymph contains lysozyme and antibacterial peptides (22). Lysozyme is mainly found in the hemolymph but has also been identified in the pericardial cells of M. sexta (7), in the gut of several insects (10) and in eggs from the cotton boll weevil (14). In hemolymph from unvaccinated H. virescens larvae, the lysozyme concentration was 6.6 mg/ml. The hemolymph lysozyme concentration greatly increased to 59.9 mg/ml after the Heliothis larvae were vaccinated with E. cloacae. A 9-fold increase in lysozyme concentration was therefore seen when comparing unvaccinated with vaccinated larvae indicating an inducible protein. Even though lysozyme is found to be ubiquitous in insects, there are nevertheless chemical and physical differences in the lysozymes isolated from different insect species within the Lepidopteran order. Lysozyme from H. virescens was compared to lysozymes from other insects. Heliothis lysozyme is more similar in molecular weight and heat sensitivity to lysozyme isolated from B. mori than to lysozyme found in H. cecropia or G. mellonella. Migration of H. virescens lysozyme on an acid gel was faster than egg white lysozyme and its molecular mass was determined to be 16,000 daltons by SDS-PAGE. Heliothis lysozyme was very resistant to heat denaturation and was stable at an acid pH. The isoelectric point of Heliothis lysozyme was estimated as greater than 9.5. This compares with the pl of H. cecropia lysozyme which was also determined to be greater than 9.5 (6). Inducible proteins like lysozyme, cecropin and attacin are well characterized proteins which have a role in defense against bacterial infections (1,2). Although lysozyme is directly active against a few Gram-positive bacteria, its main function is hydrolysis of the peptidoglycan cell wall after bacteria have already been killed by other insect defensive proteins and phagocytosis (20). Two insect immune proteins have recently been identified in larvae of H. virescens as cecropinlike and attacin-like proteins (22–25). Along with these two antibacterial proteins and the purification of lysozyme in this study, we have thereby documented the existence of the three major inducible immune proteins in H. virescens. By determining the important proteins of this insect immune system, we can better elucidate the roles of the individual proteins in the Heliothis defensive response to bacterial infections. REFERENCES 1. Dunn, P. E. (1990) BioScience 40, 738–744. 2. Boman, H. G., and Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103–126. 507
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