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Monodin-induced agglutination of Vibrio vulnificus, a major infective bacterium in black tiger prawn (Penaeus monodon)
Comp. Biochem. Physiol. Vol. 102B,No. 4, pp. 855-859, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd
MONODIN-INDUCED AGGLUTINATION OF VIBRIO VULNIFICUS, A MAJOR INFECTIVE BACTERIUM IN BLACK TIGER PRAWN (PENAEUS MONODON) SUNANTA RATANAPOand MONTRI CHULAVATNATOL* Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: (662) 2455196; Fax: (662) 2480375
(Received 3 December 1991) Abstract--1. Monodin, a sialic acid-specific lectin from P. monodon, was found to induce the agglutination of Vibrio vulnificus, a major infective bacterium of the prawn. 2. The monodin-induced agglutination of V. vulnificus was specifically inhibited by N-acetylneuraminic acid and anti-monodin. 3. During growth in a liquid medium, V. vulnificas can be induced to agglutinate only during the early stationary phase. 4. An elevation of the monodin level was found in most of the prawns suffering from bacterial infection.
INTRODUCTION
MATERIALS AND METHODS
The defense role of lectins in invertebrates against infective microbes and parasites has been a subject of interest for a long time (Anderson and Good, 1976; Ratcliffe et al., 1985; Olafsen, 1988). For example, stimulation of hemolymph lectin activity by pathogenic bacteria has been demonstrated in Pacific oyster, Crassostrea gigas (Hardy et al., 1977). Similar events have also been reported in earthworm, Lumbricus terrestris (Stein et al., 1986) and silk moth, Antheraea pernyi (Qu et al., 1987). F o r some sialic acid-specific lectins of invertebrates, limulin from American horseshoe crab, Limulus polyphemus (Robey and Liu, 1981), and carcinoscorpin from Indian horseshoe crab, Careinoscorpus rotunda cauda (Dorai et al., !982), it has been shown that they can cause agglutination of m a n y bacteria, suggesting their possible roles as anti-bacterial agents. Similarly, a sialic acidspecific lectin purified from the hemolymph of blue crab, Callinectes sapidus, has been found to possess agglutinating activity against several serotypes of Vibrio parahemolyticus (Cassels et al., 1986). Recently, we have discovered a new sialic acidspecific lectin, monodin, in the hemolymph of the black tiger prawn, Penaeus monodon (Ratanapo and Chulavatnatol, 1990). M o n o d i n has been isolated and shown to be a glycoprotein having a native molecular weight of 420,000 and consisting of subunits each with molecular weight of 27,000. It binds specifically to N-acetylneuraminic acid and requires calcium ion for its activity. This report further describes the anti-bacterial activity of m o n o d i n against a specific infective bacterium, Vibrio vulnificus of the black tiger prawn. *To whom correspondence should be addressed.
Purification of monodin Monodin was purified from the hemolymph of Penaeus monodon by affinity chromatography using a fetuin-agarose column and FPLC system (Pharmacia, Uppsala, Sweden) using a Superose-12 column as previously described (Ratanapo and Chulavatnatol, 1990). The preparation was dialyzed against 0.02 M piperazine-HC1, pH 6.0 and further purified by FPLC anion-exchange chromatography on a Mono-Q (HR 5/5) column (Pharmacia). The column was eluted with a linear gradient of 0-0.4 M NaCI. After an overnight dialysis against distilled water, the collected fractions were tested for hemagglutination activity and the active fractions were pooled, concentrated and stored for use in the experiments. Preparation of anti-monodin The Superose-12 purified lectin was applied on a thick SDS-polyacrylamide slab gel as a stripe and electrophoresed. The band of monodin subunit, with a molecular weight of 27,000 located by comparison with a stained gel, was cut and macerated before being placed in a sample cup. The monodin was then etectro-eluted at 100 V for 8 hr by using an electrophoretic sample concentrator (ISCO model 1750) filled with 0.04 M Tris-acetate, pH 8.3 containing 0.002 M EDTA. The eluted monodin was then dialyzed against 0.05 M phosphate buffer, pH 7.5 containing 0.15 M NaC1. An aliquot (20/zg protein) of the eluted monodin was emulsified with an equal volume of Freund's complete adjuvant and immunized a white rabbit. The anti-monodin was prepared and partially purified as previously described (Ratanapo and Chulavatnatol, 1990). Hemagglutination assay Hemagglutination reaction was performed in a microwell plate 96 U (Nunc) at room temperature according to Ratanapo and Chulavatnatol (1990). Hemagglutination in the presence of antibody Serial dilution of the partially purified anti-monodin was made in Tris-buffered saline, TBS (100mM Tris-HCl,
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SUNANTA RATANAPO and MONTRI CHULAVATNATOL
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Growth of V. vulnificus in liquid medium was initiated by using a single colony of V. vulnificus emulsified in 5 ml of tryptic soy broth containing 2% NaC1. The overnight bacterial culture was then inoculated into tryptic soy broth containing 2% NaC1 to give a final 0.2% inoculum and incubated at 37°C with continuous rotary shaking at 200 rpm. An aliquot (10 ml) of the bacterial culture was removed at 1 hr intervals during the growth period of 15 hr. The bacterial growth was followed by measuring the absorbance at 600 nm of the collected aliquots. The bacterial cells in each suspension were harvested, washed and resuspended to 109 cells/ml as described above.
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| ._1| 6 12 24 31 FRACTION NUMBER Fig. 1. Elution profile of monodin from a Mono-Q column. An aliquot of (1 ml) the monodin obtained from the Superose-12 column was applied to a Mono-Q column (Hr 5/5) pre-equilibrated with 20 mM piperazine-HC1, pH 6.0. After washing with the same buffer for 7 ml, a linear gradient of 0-0.4 M NaC1 (6ml in total volume) in the buffer was applied and followed by 1 M NaC1 (1 ml). The protein absorbance at 280nm (solid line) and the salt gradient (dotted line) were recorded by the FPLC system. The hemagglutination activity (HA; histogram) was assayed as described in the Materials and Methods.
1|
pH 7.5 containing 50 mM NaC1 and 10 m M CaCI2). Equal volume of the Mono-Q-purified lectin with HA activity for 2 units was then added to the dilutions, mixed and incubated for 30 min at room temperature. The hemagglutination was observed after addition of the 2% erythrocyte suspension and left for 45 min. The control test was performed in the same manner using the partially purified pre-immune serum instead of the antibody.
Preparation o f bacteria Four isolates of Vibrio vulnificus, Vibrio parahemolyticus, Vibrio angillarum and Aeromonas hydrophilla from infected P. monodon were obtained from Dr Chalor Limsuwan, Faculty of Fishery, Kasetsart University, Bangkok. Each isolate was grown on a tryptic soy agar slant containing 2% NaC1 at 37°C overnight. Other bacterial isolates including Escherichia coli, Proteus morganii, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus were obtained from the Department o f Microbiology, Mahidol University, Bangkok. Each was grown on Luria Bertani (LB) medium slant at 37°C overnight. The culture was then harvested and emulsified in TBS. The bacterial ceils were then washed thrice and resuspended in the buffer. The turbid suspensions were adjusted to approximately 109 cells/ml (A55o was equal to a No. 3 McFarland barium sulfate standard).
Bacterial agglutination test The working concentration of monodin for bacterial agglutination was determined by the method adapted from the procedure of Schaefer et al. (1979) using a suspension of V. vulnificus. The Mono-Q-purified lectin (HA = 2048 units, protein = 0.09mg/ml) was two-fold serially diluted with TBS. In each 500/11 microfuge tube, 25/~1 of the V. vulnificus suspension was mixed with 25 #1 of the diluted lectin. The tubes were left at room temperature for 10 min and was then shaken at full speed on a Vortex-Genie 2 for 20 sec. One drop of the mixture was then placed on a slide and covered with a cover-slip. The agglutination was examined under a light microscope with dark-field illumination and recorded as negative or positive ( + 1 to +4). The highest dilution of monodin giving a complete agglutination was used as the working concentration for performing subsequent agglutination tests on other bacteria. A suspension of bacteria was mixed with equal volume of the Mono-Q-purified lectin at the working concentration in 500 pl microfuge tube. To a control tube, TBS was added, instead of the monodin solution. The agglutination was carried out and rated as described above. Inhibition o f bacterial agglutination by sugar and antimonodin To test the sugar inhibition of the monodin-induced agglutination of 11".vulnificus, the selected sugar was twofold serially diluted with TBS. The Mono Q-purified monodin of twice the working concentration (25#1) was mixed with 25/~1 of the diluted sugar. After incubation at room temperature for 10min, 25pl of the V. vulnificus suspension was added and the mixture was further left standing for an additional 10min. The inhibition was observed under a light microscope and rated as above. The minimal concentration of the sugar required for the complete inhibition was recorded. To test the inhibition by anti-monodin, the above was performed using anti-monodin instead of the sugar. However, the incubation time between the monodin and the antibody was carried out for 30 min. Protein determination Concentration of protein samples was determined by the method of Lowry et al. (1951). SDS-polyacrylamide gel electrophoresis Slab gel electrophoresis was performed by the method of Laemmli (1970), using a 7-15% gradient polyacrylamide gel
Table 1. Purification of monodin from the hemolymph of P. monodon
Fraction
Volume (ml)
Protein (mg)
Total HA activity (units)
Specific activity (units/mg)
Purification (-fold)
Recovery (%)
Serum 33.6 4,500 362,496 81 1 100 Affinity-purified lectin 5.1 0.612 52,224 85,333 1,053 14.4 Superose 12-purified lectin 5.3 0.048 13,515 281,563 3,476 3.7 Mono Q-purified lectin 2.6 0.031 13,107 422,820 5,220 3.6 The units of hemagglutination activity were reported as the reciprocal of the highest dilution of lectin giving complete agglutination after 45 min incubation with the 2% human erythrocyte suspension.
Monodin-induced bacterial agglutination
A
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B
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Fig. 2. Agglutination of V. vulnificus induced by monodin. The bacterial sample was incubated in TBS (A) and with monodin (B). slab in the presence of 0.1% sodium dodecylsulfate (SDS). Molecular weight markers used were phosphorylase b (94,000), bovine serum albumin (68,000), ovalbumin (43,000) and cytochrome c (12,500).
Immunoblotting detection of monodin Protein bands separated by SDS-PAGE were electrophoretically blotted onto a nitrocellulose sheet according to the method of Gershoni and Palade (1983). The gel was pre-swollen for 30 min in the transferred buffer (0.025 M Tris, 0.192 M glycine, 20% (v/v) methanol, pH 8.3). The proteins in the gel were transferred to the nitrocellulose sheet at room temperature in a Hoeffer transfer apparatus operating at 0.5 mA for 2hr. After treating the nitrocellulose blot with 5% skim milk in PBS (0.02 M phosphate buffer, pH 7.4, containing 0.15 M NaC1) for 2 hr at room temperature, immunostaining of the blot was performed by incubating the blot for 2 hr with a 2000-fold dilution of either the partially purified anti-monodin (primary antibody) or pre-immunized serum. The blot was washed thrice for 10min each with PBS containing 0.1% Tween-20, followed by incubation with 1000-fold diluted alkaline phosphatase conjugated anti-IgG (secondary antibody) for 2 hr. After washing once with PBS containing 0.1% Tween20 and thrice with PBS, the purple band of the monodin was visualized 4 rain after adding a substrate solution (0.03 g nitroblue tetrazolium in 1 ml of 70% dimethylsulfoxide, 0.015 g 5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt in 1 ml of 100% dimethylsulfoxide and 98 ml of 1 M carbonate buffer, pH 9.8 containing 1 mM MgC12). Table 2. Agglutinationof V. vulnificusinducedby monodin and other lectins Minimum concentration requires Lectins (ug/ml) Monodin Lumulin Wheat germ agglutinin Limax flavus agglutinin Potato lectin Phaseolus vulgaris agglutinin-L
2.8 15.6 125 156 250 1000
RESULTS
Further purification of monodin To clearly establish the biological role of monodin, it was desirable to have a highly purified lectin for critical work. When the Superose-12-purified m o n o d i n prepared as described previously (Ratanapo and Chulavatnatol, 1990) was further purified using a M o n o - Q column, two main protein peaks were obtained (Fig. 1). The lectin activity was found to be associated with only one peak, eluted at 0.37 M NaC1. The purity of m o n o d i n was much improved as a result of using the M o n o Q column (Table 1).
Agglutination of Vibrio vulnificus Of several bacteria isolated from infected P.
monodon, three were identified to be Vibrio vulnificus, Vibrio parahemolyticus and Vibrio anguillarum. A m o n g these, V. vulnificus was the predominant species. To elucidate the possible role of m o n o d i n in the infected prawns, the possible monodin-induced agglutination of these pathogenic bacteria was investigated. Using the highly purified m o n o d i n from the M o n o - Q column (2.8/ag/ml), V. vulnificus was found to be agglutinated (Fig. 2) while the other pathogenic bacteria of the prawn were not. Under the same condition, the Mono-Q-purified m o n o d i n did not cause any agglutination of six other bacteria tested, namely Aeromonas hydrophilla, Escherichia coli,
Proteus morganii, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus. Characteristics of the monodin-induced agglutination of V. vulnificus The interaction between the prawn lectin, monodin, and the major infective bacterium, V. vulnificus, of the prawn was further studied. The following results were obtained.
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Fig. 3. The relationship between the growth of V. vulnificus in liquid medium and the monodin-induced agglutination (indicated by pluses). (a) Sugar inhibition. The sugar specificity of the interaction between monodin and V. vulnificus was investigated by performing the sugar inhibition test. Among nine monosaccharides tested, only N-acetylneuraminic acid at 12.5mM was found to be fully inhibitory to the monodin-induced agglutination of the bacteria. Others which were non-inhibitory at 50mM were N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, galactose, glucose, glucosamine, mannose and fucose. (b) Inhibition by anti-monodin. The activity of monodin to induce the agglutination of V. vulnificus was found to be neutralized by the rabbit antimonodin. Complete inhibition required 20/tg/ml of the anti-monodin. (c) Specificity for monodin. Comparison was made between monodin and other lectins in causing the
A B
C
D
E
CHULAVATNATOL
V. vulnificus agglutination. Among several lectins tested, monodin was found to be most effective. Five other lectins were also found to possess the ability to induce the V. vulnificus agglutination but they did so at the concentrations higher than that of monodin (Table 2). In decreasing order of effectiveness, they were limulin, Limax flavus agglutinin, wheat germ agglutinin, potato lectin and Phaseolus vulgaris agglutinin-L. Other lectins which were found to be inactive at 1 mg/ml were jacalin, jackfruit lectin, Concanavalin A, lentil lectin, Banadeiraea simplicifolia lectins I and II, peanut lectin, Maclura pomifera lectin, horse gram lectin, Bauhinia purpurea lectin, Roman snail lectin, soybean lectin and Phaseolus vulgaris agglutinin-E. (d) Dependence on the bacterial growth. The monodin-induced agglutination of V. vulnificus during its growth in a liquid medium was studied. Under the culture condition used, the bacteria during the lag and log phases could not be induced to agglutinate by monodin. However, the agglutination induced by monodin was observed only during the early stationary phase (Fig. 3) which covered the period of 6-9 hr of growth in the liquid medium. After that, the agglutination could not be induced by monodin. Compar&on of the monodin levels between the infected and uninfected prawns P. monodon, which suffered from bacterial infection, showed discoloration of the body tissue or loss of some anatomical parts. Samples of the bacteriainfected P. monodon were collected from a local farm and the hemolymph samples were collected. Of the 35 infected prawns, 74% showed slow or no clotting of the hemolymph. In this experiment, sera from the clotted hemolymph of 7 infected male prawns of 90-days-old were used. The serum protein of the infected samples (346.8 ___57.9 mg/ml) was about 1.4
F
G
H
I
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............l Fig. 4. Immunoblotting of the sera of infected P. monodon. Lane A: purified monodin (9/tg); lanes B, C: sera of uninfected prawns; and lanes D-J: sera of infected prawns. The arrow indicates the position of the band reactive to anti-monodin and having the molecular weight of 27,000.
859
Monodin-induced bacterial agglutination times higher than that (125.2 + 1.5 mg/mi) of the uninfected prawns of the same age. By S D S polyacrylamide gel electrophoresis and immunoblotting, using anti-monodin as the primary antibody, sera of 5 out of 7 infected prawns showed elevated levels of monodin in comparison to those of the uninfected prawns (Fig. 4). DISCUSSION The results described in this report strongly support our contention that monodin, which is a sialic acid-specific lectin in the hemolymph of P. monodon, plays a role in the defense against the major infective bacterium, V. vulnificus, of the prawn. The monodininduced agglutination of the bacterium may be similar to the roles of humoral substances that agglutinate foreign agents in other invertebrates (Ratcliffe et al., 1985). The specific inhibition of the agglutination by N-acetylneuraminic acid suggests that the monodin may bind with N-acetylneuraminic acid on the bacterial surface. This suggestion is further strengthened by the observation that other lectins with specificity toward sialic acids, namely limulin and wheat germ agglutinin (Table 2) are also partially inhibitory. The sugar residue which serves as the monodinbinding site on the surface of V. vulnificus is apparently exposed or exists for a limited period during the early stationary phase (Fig. 3). The observation implies that the bacterial surface undergoes dynamic changes during growth. This may be a mechanism by which the bacterium tries to escape the defense of the prawn. The elevation of the monodin level in the infected prawn (Fig. 4) implies that the defensive lectin may be inducible by the infective V. vulnificus. However, since the increase in the monodin level is not found in all infected prawns studied, the lectin induction may be under some unidentified control. In most invertebrates, lectins are not induced by antigenic stimulation (Ratcliffe et al., 1985). However, hemolymph lectin induction by foreign particles has been observed in blue crab, Callinectes sapidus (Pauley, 1973). In oyster, Crassostrea gigas, the lectin level increases after a challenge by Vibrio anguillarum (Hardy et al., 1977). Therefore, the induction of monodin should be further investigated by a direct challenge with V. vulnificus. In this study, use of the highly purified monodin (Table 1) should minimize any chance of error in the conclusion due to the presence of any impurity in the lectin preparation. Furthermore, the inhibition of
the monodin-induced bacterial agglutination by antimonodin should be an additional assurance that the agglutination is due to the lectin itself. REFERENCES
Anderson R. S. and Good R. A. (1976) Opsonic involvement in phagocytosis by mollusk hemocytes. J. Invertebr. Pathol. 27, 57-64. Cassells F. J., Marchalonis J. J. and Vasta G. R. (1986) Heterogeneous humoral and hemocyte associated lectins with N-acylaminosugar specificities from the blue crab, Callinectes sapidus rathbun. Comp. Biochem. Physiol. 85B, 23-30. Dorai D. T., Mohan S., Srimal S. and Bacchawat B. K. (1982) On the multispecificity of carcinoscorpin, the sialic acid binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda: recognition of glycerophosphate in membrane teichoic acid. FEBS Lett. 148, 98-102. Gershoni J. M. and Palade G. E. (1983) Protein blotting: principles and applications. Analyt. Biochem. 131, 1-15. Hardy S. W., Grant P. T. and Fletcher T. C. (1977) A hemagglutinin in the tissue fluid of the Pacific oyster, Crassostrea gigas with specificity for sialic acid residues in glycoproteins. Experientia 33, 767-769. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227, 680~85. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. Olafsen J. A. (1988) Role of lectins in invertebrate humoral defense. Am. Fish. Soc. Spec. Publ. 18, 189-205. Pauley G. B. (1973) An attempt to immunize the blue crab, Callinectes sapidus, with vertebrate red blood cells. Experientia 29, 210-21 I. Qu X. M., Zhang C. F., Komano H. and Natori S. (1987) Purification of a lectin from the hemolymph of Chinese oak silk moth (Antheraeapernyi) pupae. J. Biochem. I01, 545-551. Ratanapo S. and Chulavatnatol M. (1990) Monodin, a new sialic acid-specific lectin from black tiger prawn (Penaeus monodon). Comp. Biochem. Physiol. 97B, 515-520. Ratcliffe N. A., Rowley A. F., Fitzgerald S. W. and Rhodes C. P. (1985) Invertebrate immunity: Basic concepts and recent advances. Int. Rev. Cytol. 97, 183-350. Robey F. A. and Liu T. Y. (1981) Limulin: a C-reactive protein from Limulus polyphemus. J. biol. Chem. 256, 969-975. Schaefer R. L., Keller K. F. and Doyle R. J. (1979) Lectins in diagnostic microbiology: use of wheat germ agglutinin for laboratory identification of Neisseria gonorrhoeae. J. clin. Microbiol. I0, 669~72. Stein E. A., Soheil Y. and Cooper E. L. (1986) Bacterial agglutinins of the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. 84B, 409-415.
0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd
MONODIN-INDUCED AGGLUTINATION OF VIBRIO VULNIFICUS, A MAJOR INFECTIVE BACTERIUM IN BLACK TIGER PRAWN (PENAEUS MONODON) SUNANTA RATANAPOand MONTRI CHULAVATNATOL* Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: (662) 2455196; Fax: (662) 2480375
(Received 3 December 1991) Abstract--1. Monodin, a sialic acid-specific lectin from P. monodon, was found to induce the agglutination of Vibrio vulnificus, a major infective bacterium of the prawn. 2. The monodin-induced agglutination of V. vulnificus was specifically inhibited by N-acetylneuraminic acid and anti-monodin. 3. During growth in a liquid medium, V. vulnificas can be induced to agglutinate only during the early stationary phase. 4. An elevation of the monodin level was found in most of the prawns suffering from bacterial infection.
INTRODUCTION
MATERIALS AND METHODS
The defense role of lectins in invertebrates against infective microbes and parasites has been a subject of interest for a long time (Anderson and Good, 1976; Ratcliffe et al., 1985; Olafsen, 1988). For example, stimulation of hemolymph lectin activity by pathogenic bacteria has been demonstrated in Pacific oyster, Crassostrea gigas (Hardy et al., 1977). Similar events have also been reported in earthworm, Lumbricus terrestris (Stein et al., 1986) and silk moth, Antheraea pernyi (Qu et al., 1987). F o r some sialic acid-specific lectins of invertebrates, limulin from American horseshoe crab, Limulus polyphemus (Robey and Liu, 1981), and carcinoscorpin from Indian horseshoe crab, Careinoscorpus rotunda cauda (Dorai et al., !982), it has been shown that they can cause agglutination of m a n y bacteria, suggesting their possible roles as anti-bacterial agents. Similarly, a sialic acidspecific lectin purified from the hemolymph of blue crab, Callinectes sapidus, has been found to possess agglutinating activity against several serotypes of Vibrio parahemolyticus (Cassels et al., 1986). Recently, we have discovered a new sialic acidspecific lectin, monodin, in the hemolymph of the black tiger prawn, Penaeus monodon (Ratanapo and Chulavatnatol, 1990). M o n o d i n has been isolated and shown to be a glycoprotein having a native molecular weight of 420,000 and consisting of subunits each with molecular weight of 27,000. It binds specifically to N-acetylneuraminic acid and requires calcium ion for its activity. This report further describes the anti-bacterial activity of m o n o d i n against a specific infective bacterium, Vibrio vulnificus of the black tiger prawn. *To whom correspondence should be addressed.
Purification of monodin Monodin was purified from the hemolymph of Penaeus monodon by affinity chromatography using a fetuin-agarose column and FPLC system (Pharmacia, Uppsala, Sweden) using a Superose-12 column as previously described (Ratanapo and Chulavatnatol, 1990). The preparation was dialyzed against 0.02 M piperazine-HC1, pH 6.0 and further purified by FPLC anion-exchange chromatography on a Mono-Q (HR 5/5) column (Pharmacia). The column was eluted with a linear gradient of 0-0.4 M NaCI. After an overnight dialysis against distilled water, the collected fractions were tested for hemagglutination activity and the active fractions were pooled, concentrated and stored for use in the experiments. Preparation of anti-monodin The Superose-12 purified lectin was applied on a thick SDS-polyacrylamide slab gel as a stripe and electrophoresed. The band of monodin subunit, with a molecular weight of 27,000 located by comparison with a stained gel, was cut and macerated before being placed in a sample cup. The monodin was then etectro-eluted at 100 V for 8 hr by using an electrophoretic sample concentrator (ISCO model 1750) filled with 0.04 M Tris-acetate, pH 8.3 containing 0.002 M EDTA. The eluted monodin was then dialyzed against 0.05 M phosphate buffer, pH 7.5 containing 0.15 M NaC1. An aliquot (20/zg protein) of the eluted monodin was emulsified with an equal volume of Freund's complete adjuvant and immunized a white rabbit. The anti-monodin was prepared and partially purified as previously described (Ratanapo and Chulavatnatol, 1990). Hemagglutination assay Hemagglutination reaction was performed in a microwell plate 96 U (Nunc) at room temperature according to Ratanapo and Chulavatnatol (1990). Hemagglutination in the presence of antibody Serial dilution of the partially purified anti-monodin was made in Tris-buffered saline, TBS (100mM Tris-HCl,
855
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SUNANTA RATANAPO and MONTRI CHULAVATNATOL
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Growth of V. vulnificus in liquid medium was initiated by using a single colony of V. vulnificus emulsified in 5 ml of tryptic soy broth containing 2% NaC1. The overnight bacterial culture was then inoculated into tryptic soy broth containing 2% NaC1 to give a final 0.2% inoculum and incubated at 37°C with continuous rotary shaking at 200 rpm. An aliquot (10 ml) of the bacterial culture was removed at 1 hr intervals during the growth period of 15 hr. The bacterial growth was followed by measuring the absorbance at 600 nm of the collected aliquots. The bacterial cells in each suspension were harvested, washed and resuspended to 109 cells/ml as described above.
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| ._1| 6 12 24 31 FRACTION NUMBER Fig. 1. Elution profile of monodin from a Mono-Q column. An aliquot of (1 ml) the monodin obtained from the Superose-12 column was applied to a Mono-Q column (Hr 5/5) pre-equilibrated with 20 mM piperazine-HC1, pH 6.0. After washing with the same buffer for 7 ml, a linear gradient of 0-0.4 M NaC1 (6ml in total volume) in the buffer was applied and followed by 1 M NaC1 (1 ml). The protein absorbance at 280nm (solid line) and the salt gradient (dotted line) were recorded by the FPLC system. The hemagglutination activity (HA; histogram) was assayed as described in the Materials and Methods.
1|
pH 7.5 containing 50 mM NaC1 and 10 m M CaCI2). Equal volume of the Mono-Q-purified lectin with HA activity for 2 units was then added to the dilutions, mixed and incubated for 30 min at room temperature. The hemagglutination was observed after addition of the 2% erythrocyte suspension and left for 45 min. The control test was performed in the same manner using the partially purified pre-immune serum instead of the antibody.
Preparation o f bacteria Four isolates of Vibrio vulnificus, Vibrio parahemolyticus, Vibrio angillarum and Aeromonas hydrophilla from infected P. monodon were obtained from Dr Chalor Limsuwan, Faculty of Fishery, Kasetsart University, Bangkok. Each isolate was grown on a tryptic soy agar slant containing 2% NaC1 at 37°C overnight. Other bacterial isolates including Escherichia coli, Proteus morganii, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus were obtained from the Department o f Microbiology, Mahidol University, Bangkok. Each was grown on Luria Bertani (LB) medium slant at 37°C overnight. The culture was then harvested and emulsified in TBS. The bacterial ceils were then washed thrice and resuspended in the buffer. The turbid suspensions were adjusted to approximately 109 cells/ml (A55o was equal to a No. 3 McFarland barium sulfate standard).
Bacterial agglutination test The working concentration of monodin for bacterial agglutination was determined by the method adapted from the procedure of Schaefer et al. (1979) using a suspension of V. vulnificus. The Mono-Q-purified lectin (HA = 2048 units, protein = 0.09mg/ml) was two-fold serially diluted with TBS. In each 500/11 microfuge tube, 25/~1 of the V. vulnificus suspension was mixed with 25 #1 of the diluted lectin. The tubes were left at room temperature for 10 min and was then shaken at full speed on a Vortex-Genie 2 for 20 sec. One drop of the mixture was then placed on a slide and covered with a cover-slip. The agglutination was examined under a light microscope with dark-field illumination and recorded as negative or positive ( + 1 to +4). The highest dilution of monodin giving a complete agglutination was used as the working concentration for performing subsequent agglutination tests on other bacteria. A suspension of bacteria was mixed with equal volume of the Mono-Q-purified lectin at the working concentration in 500 pl microfuge tube. To a control tube, TBS was added, instead of the monodin solution. The agglutination was carried out and rated as described above. Inhibition o f bacterial agglutination by sugar and antimonodin To test the sugar inhibition of the monodin-induced agglutination of 11".vulnificus, the selected sugar was twofold serially diluted with TBS. The Mono Q-purified monodin of twice the working concentration (25#1) was mixed with 25/~1 of the diluted sugar. After incubation at room temperature for 10min, 25pl of the V. vulnificus suspension was added and the mixture was further left standing for an additional 10min. The inhibition was observed under a light microscope and rated as above. The minimal concentration of the sugar required for the complete inhibition was recorded. To test the inhibition by anti-monodin, the above was performed using anti-monodin instead of the sugar. However, the incubation time between the monodin and the antibody was carried out for 30 min. Protein determination Concentration of protein samples was determined by the method of Lowry et al. (1951). SDS-polyacrylamide gel electrophoresis Slab gel electrophoresis was performed by the method of Laemmli (1970), using a 7-15% gradient polyacrylamide gel
Table 1. Purification of monodin from the hemolymph of P. monodon
Fraction
Volume (ml)
Protein (mg)
Total HA activity (units)
Specific activity (units/mg)
Purification (-fold)
Recovery (%)
Serum 33.6 4,500 362,496 81 1 100 Affinity-purified lectin 5.1 0.612 52,224 85,333 1,053 14.4 Superose 12-purified lectin 5.3 0.048 13,515 281,563 3,476 3.7 Mono Q-purified lectin 2.6 0.031 13,107 422,820 5,220 3.6 The units of hemagglutination activity were reported as the reciprocal of the highest dilution of lectin giving complete agglutination after 45 min incubation with the 2% human erythrocyte suspension.
Monodin-induced bacterial agglutination
A
857
B
g
1 1
Fig. 2. Agglutination of V. vulnificus induced by monodin. The bacterial sample was incubated in TBS (A) and with monodin (B). slab in the presence of 0.1% sodium dodecylsulfate (SDS). Molecular weight markers used were phosphorylase b (94,000), bovine serum albumin (68,000), ovalbumin (43,000) and cytochrome c (12,500).
Immunoblotting detection of monodin Protein bands separated by SDS-PAGE were electrophoretically blotted onto a nitrocellulose sheet according to the method of Gershoni and Palade (1983). The gel was pre-swollen for 30 min in the transferred buffer (0.025 M Tris, 0.192 M glycine, 20% (v/v) methanol, pH 8.3). The proteins in the gel were transferred to the nitrocellulose sheet at room temperature in a Hoeffer transfer apparatus operating at 0.5 mA for 2hr. After treating the nitrocellulose blot with 5% skim milk in PBS (0.02 M phosphate buffer, pH 7.4, containing 0.15 M NaC1) for 2 hr at room temperature, immunostaining of the blot was performed by incubating the blot for 2 hr with a 2000-fold dilution of either the partially purified anti-monodin (primary antibody) or pre-immunized serum. The blot was washed thrice for 10min each with PBS containing 0.1% Tween-20, followed by incubation with 1000-fold diluted alkaline phosphatase conjugated anti-IgG (secondary antibody) for 2 hr. After washing once with PBS containing 0.1% Tween20 and thrice with PBS, the purple band of the monodin was visualized 4 rain after adding a substrate solution (0.03 g nitroblue tetrazolium in 1 ml of 70% dimethylsulfoxide, 0.015 g 5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt in 1 ml of 100% dimethylsulfoxide and 98 ml of 1 M carbonate buffer, pH 9.8 containing 1 mM MgC12). Table 2. Agglutinationof V. vulnificusinducedby monodin and other lectins Minimum concentration requires Lectins (ug/ml) Monodin Lumulin Wheat germ agglutinin Limax flavus agglutinin Potato lectin Phaseolus vulgaris agglutinin-L
2.8 15.6 125 156 250 1000
RESULTS
Further purification of monodin To clearly establish the biological role of monodin, it was desirable to have a highly purified lectin for critical work. When the Superose-12-purified m o n o d i n prepared as described previously (Ratanapo and Chulavatnatol, 1990) was further purified using a M o n o - Q column, two main protein peaks were obtained (Fig. 1). The lectin activity was found to be associated with only one peak, eluted at 0.37 M NaC1. The purity of m o n o d i n was much improved as a result of using the M o n o Q column (Table 1).
Agglutination of Vibrio vulnificus Of several bacteria isolated from infected P.
monodon, three were identified to be Vibrio vulnificus, Vibrio parahemolyticus and Vibrio anguillarum. A m o n g these, V. vulnificus was the predominant species. To elucidate the possible role of m o n o d i n in the infected prawns, the possible monodin-induced agglutination of these pathogenic bacteria was investigated. Using the highly purified m o n o d i n from the M o n o - Q column (2.8/ag/ml), V. vulnificus was found to be agglutinated (Fig. 2) while the other pathogenic bacteria of the prawn were not. Under the same condition, the Mono-Q-purified m o n o d i n did not cause any agglutination of six other bacteria tested, namely Aeromonas hydrophilla, Escherichia coli,
Proteus morganii, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus. Characteristics of the monodin-induced agglutination of V. vulnificus The interaction between the prawn lectin, monodin, and the major infective bacterium, V. vulnificus, of the prawn was further studied. The following results were obtained.
858
SUNANTARATANAPOand MONTRI AGGLUTINATION .
.
.
.
.
.
++ +÷ +4
+
i.5
+
.
.
.
.
.
++
x .J .-I l,IJ
0.s LI. o
1.1.1
:E --i Z
0.1 _
I 2
4
|
|
ll
I
I
12
14
TIME (HOUR)
Fig. 3. The relationship between the growth of V. vulnificus in liquid medium and the monodin-induced agglutination (indicated by pluses). (a) Sugar inhibition. The sugar specificity of the interaction between monodin and V. vulnificus was investigated by performing the sugar inhibition test. Among nine monosaccharides tested, only N-acetylneuraminic acid at 12.5mM was found to be fully inhibitory to the monodin-induced agglutination of the bacteria. Others which were non-inhibitory at 50mM were N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, galactose, glucose, glucosamine, mannose and fucose. (b) Inhibition by anti-monodin. The activity of monodin to induce the agglutination of V. vulnificus was found to be neutralized by the rabbit antimonodin. Complete inhibition required 20/tg/ml of the anti-monodin. (c) Specificity for monodin. Comparison was made between monodin and other lectins in causing the
A B
C
D
E
CHULAVATNATOL
V. vulnificus agglutination. Among several lectins tested, monodin was found to be most effective. Five other lectins were also found to possess the ability to induce the V. vulnificus agglutination but they did so at the concentrations higher than that of monodin (Table 2). In decreasing order of effectiveness, they were limulin, Limax flavus agglutinin, wheat germ agglutinin, potato lectin and Phaseolus vulgaris agglutinin-L. Other lectins which were found to be inactive at 1 mg/ml were jacalin, jackfruit lectin, Concanavalin A, lentil lectin, Banadeiraea simplicifolia lectins I and II, peanut lectin, Maclura pomifera lectin, horse gram lectin, Bauhinia purpurea lectin, Roman snail lectin, soybean lectin and Phaseolus vulgaris agglutinin-E. (d) Dependence on the bacterial growth. The monodin-induced agglutination of V. vulnificus during its growth in a liquid medium was studied. Under the culture condition used, the bacteria during the lag and log phases could not be induced to agglutinate by monodin. However, the agglutination induced by monodin was observed only during the early stationary phase (Fig. 3) which covered the period of 6-9 hr of growth in the liquid medium. After that, the agglutination could not be induced by monodin. Compar&on of the monodin levels between the infected and uninfected prawns P. monodon, which suffered from bacterial infection, showed discoloration of the body tissue or loss of some anatomical parts. Samples of the bacteriainfected P. monodon were collected from a local farm and the hemolymph samples were collected. Of the 35 infected prawns, 74% showed slow or no clotting of the hemolymph. In this experiment, sera from the clotted hemolymph of 7 infected male prawns of 90-days-old were used. The serum protein of the infected samples (346.8 ___57.9 mg/ml) was about 1.4
F
G
H
I
J
............l Fig. 4. Immunoblotting of the sera of infected P. monodon. Lane A: purified monodin (9/tg); lanes B, C: sera of uninfected prawns; and lanes D-J: sera of infected prawns. The arrow indicates the position of the band reactive to anti-monodin and having the molecular weight of 27,000.
859
Monodin-induced bacterial agglutination times higher than that (125.2 + 1.5 mg/mi) of the uninfected prawns of the same age. By S D S polyacrylamide gel electrophoresis and immunoblotting, using anti-monodin as the primary antibody, sera of 5 out of 7 infected prawns showed elevated levels of monodin in comparison to those of the uninfected prawns (Fig. 4). DISCUSSION The results described in this report strongly support our contention that monodin, which is a sialic acid-specific lectin in the hemolymph of P. monodon, plays a role in the defense against the major infective bacterium, V. vulnificus, of the prawn. The monodininduced agglutination of the bacterium may be similar to the roles of humoral substances that agglutinate foreign agents in other invertebrates (Ratcliffe et al., 1985). The specific inhibition of the agglutination by N-acetylneuraminic acid suggests that the monodin may bind with N-acetylneuraminic acid on the bacterial surface. This suggestion is further strengthened by the observation that other lectins with specificity toward sialic acids, namely limulin and wheat germ agglutinin (Table 2) are also partially inhibitory. The sugar residue which serves as the monodinbinding site on the surface of V. vulnificus is apparently exposed or exists for a limited period during the early stationary phase (Fig. 3). The observation implies that the bacterial surface undergoes dynamic changes during growth. This may be a mechanism by which the bacterium tries to escape the defense of the prawn. The elevation of the monodin level in the infected prawn (Fig. 4) implies that the defensive lectin may be inducible by the infective V. vulnificus. However, since the increase in the monodin level is not found in all infected prawns studied, the lectin induction may be under some unidentified control. In most invertebrates, lectins are not induced by antigenic stimulation (Ratcliffe et al., 1985). However, hemolymph lectin induction by foreign particles has been observed in blue crab, Callinectes sapidus (Pauley, 1973). In oyster, Crassostrea gigas, the lectin level increases after a challenge by Vibrio anguillarum (Hardy et al., 1977). Therefore, the induction of monodin should be further investigated by a direct challenge with V. vulnificus. In this study, use of the highly purified monodin (Table 1) should minimize any chance of error in the conclusion due to the presence of any impurity in the lectin preparation. Furthermore, the inhibition of
the monodin-induced bacterial agglutination by antimonodin should be an additional assurance that the agglutination is due to the lectin itself. REFERENCES
Anderson R. S. and Good R. A. (1976) Opsonic involvement in phagocytosis by mollusk hemocytes. J. Invertebr. Pathol. 27, 57-64. Cassells F. J., Marchalonis J. J. and Vasta G. R. (1986) Heterogeneous humoral and hemocyte associated lectins with N-acylaminosugar specificities from the blue crab, Callinectes sapidus rathbun. Comp. Biochem. Physiol. 85B, 23-30. Dorai D. T., Mohan S., Srimal S. and Bacchawat B. K. (1982) On the multispecificity of carcinoscorpin, the sialic acid binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda: recognition of glycerophosphate in membrane teichoic acid. FEBS Lett. 148, 98-102. Gershoni J. M. and Palade G. E. (1983) Protein blotting: principles and applications. Analyt. Biochem. 131, 1-15. Hardy S. W., Grant P. T. and Fletcher T. C. (1977) A hemagglutinin in the tissue fluid of the Pacific oyster, Crassostrea gigas with specificity for sialic acid residues in glycoproteins. Experientia 33, 767-769. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227, 680~85. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. Olafsen J. A. (1988) Role of lectins in invertebrate humoral defense. Am. Fish. Soc. Spec. Publ. 18, 189-205. Pauley G. B. (1973) An attempt to immunize the blue crab, Callinectes sapidus, with vertebrate red blood cells. Experientia 29, 210-21 I. Qu X. M., Zhang C. F., Komano H. and Natori S. (1987) Purification of a lectin from the hemolymph of Chinese oak silk moth (Antheraeapernyi) pupae. J. Biochem. I01, 545-551. Ratanapo S. and Chulavatnatol M. (1990) Monodin, a new sialic acid-specific lectin from black tiger prawn (Penaeus monodon). Comp. Biochem. Physiol. 97B, 515-520. Ratcliffe N. A., Rowley A. F., Fitzgerald S. W. and Rhodes C. P. (1985) Invertebrate immunity: Basic concepts and recent advances. Int. Rev. Cytol. 97, 183-350. Robey F. A. and Liu T. Y. (1981) Limulin: a C-reactive protein from Limulus polyphemus. J. biol. Chem. 256, 969-975. Schaefer R. L., Keller K. F. and Doyle R. J. (1979) Lectins in diagnostic microbiology: use of wheat germ agglutinin for laboratory identification of Neisseria gonorrhoeae. J. clin. Microbiol. I0, 669~72. Stein E. A., Soheil Y. and Cooper E. L. (1986) Bacterial agglutinins of the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. 84B, 409-415.