- Email: [email protected]
[27] Aerolysin from Aeromonas hydrophila
AEROLYSINFROMA. hydrophila
[27]
193
examination shows erosive lesions and desquamation of necrotic mucosa accompanied by marked neutrophil infiltration into the intestinal wall. Low doses of the thermostable direct hemolysin rapidly kill various experimental animals, such as mice, rats, and guinea pigs. For example, intravenous injection of 5/xg of the hemolysin killed mice within 1 min. The rapid death of the animals was shown to be due to the cardiotoxicity of the thermostable direct hemolysin. 4,1° ~0 K. Goshima, T. Honda, M. Hirata, K. Kikuchi, Y. Takeda, and T. Miwatani, J. Mol. Cell. Cardiol. 9, 191 (1977).
[27] A e r o l y s i n f r o m A e r o m o n a s hydrophila
By J. THOMAS BVCKLEY and S. PETER HOWARD Aerolysin is a soluble cytolytic protein produced by the gram-negative bacteria Aeromonas hydrophila.l,2 It is released to the extracellular medium as an inactive protoxin (proaerolysin) which is converted to the active protein by one or more extracellular proteases which are also exported by A. hydrophila. 3 The mechanism of action of the toxin is very similar to the mechanism proposed for staphylococcal alpha toxin. 4 The toxin binds to a specific glycoprotein receptor on the surface of eukaryotic cells, inserts into the lipid bilayer, and forms holes approximately 3 nm in diameter. This results in destruction of the membrane permeability barrier and cell death? Assay Method
Principle. By far the easiest methods of measuring the activity of aerolysin are based on the ability of the toxin to cause the hemolysis of red cells. Plate assays measure the clearing of red cell suspensions in wells and the absence of cell pellets at the bottom of wells. For convenience, human red cells obtained from outdated blood are normally used; F. H. Castelitz and R. Gunther, Zentralbl. Bakteriol., Parasitenk. Infektionskr. Hyg. Abt. 1: Orig. 180, 30 (1960). B. Wretlind, R. MOllby, and T. WadstrOm, Infect. lmrnun. 4, 503 (1971). 3 S. P. Howard and J. T. Buckley, J. Bacteriol. 163, 336 (1985). 4 S. Harshman, Mol. Cell. Biochem. 23, 143 (1979). 5 S. P. Howard and J. T. Buckley, Biochemistry 21, 1662 (1982).
METHODS IN ENZYMOLOGY, VOL. 165
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
194
PREPARATION OF TOXINS
[27]
however, sensitivity can be increased more than 100-fold by using rat erythrocytes.6
Reagents PBS-albumin: 10 mM taining 0.1% bovine Washed erythrocytes: man blood, washed PBS-albumin
sodium phosphate, 150 mM NaC1, pH 7.4, conserum albumin Packed human erythrocytes from outdated hu3 × with PBS and resuspended to 0.8% (v/v) in
Assay of the Active Toxin. Hemolytic activity determinations are carried out in microtiter plates after 2-fold serial dilutions of samples in 0.1 ml of PBS-albumin. An equal volume of the 0.8% erythrocytes in PBSalbumin is added to each well and the plates are incubated at 37° for 1 hr. Hemolytic activity is expressed as the inverse of the largest dilution at which complete lysis occurs. Assay of the Inactive Protoxin. The assay procedure is essentially the same as the procedure for the mature protein. The protoxin is activated in the first well of the titer plate, which for this purpose contains PBS but no albumin. Activation is achieved by adding trypsin to a final concentration of 0.1-1 /xg/ml and incubating at room temperature for 5 min. Serial dilutions into PBS-albumin are performed and the assay continued as above. The presence of trypsin inhibitor at a final concentration of 1 mg/ml in the PBS-albumin used for serial dilution has no affect on the results of the assay. Enzyme Production and Isolation
Medium. Aeromonas hydrophila is grown in 2-liter flasks, each containing 200 ml of medium prepared by dissolving 8 g of yeast extract (Difco) and 4 g of casamino acids (Difco) in a total of 156 ml of water containing 0.2 ml of 3.3 x 10-3% thiamin and 0.2 ml of 0.12% nicotinic acid stock and adjusting the pH to 7.2. Yeast RNA (20 ml of a 10% solution, pH 7.2) is also added to each flask followed by 2 ml of 0.1 M MgSO4, 2 ml of 0.01 M CaCI2, and 20 ml 10× M9 salts (60.34 g Na2HPO4, I0.19 g NH4C1, 30.4 g KH2PO4, and 5.19 g of NaCI dissolved in water to 1 liter). The hemolytic activity of extracellular culture supernatants is far higher when RNA is present in the culture medium. 7 The reason for this has not been established, but may be due to the ability of the RNA to 6 A. W. Bernheimer, L. S. Avigad, and G. Avigad, Infect. Immun. 11, 1312 (1975). 7 A. W. Bernheimer and L. S. Avigad, Infect. Imrnun. 9, 1016 (1974).
[27]
AEROLYSINFROMA. hydrophila
195
complex divalent cations and thereby inhibit one or more extracellular proteases. Growth Conditions for the Isolation of Mature Active Toxin. 8 The flasks are inoculated with 1% of an overnight culture containing 2 × 10 9 to 5 X 10 9 viable cells per milliliter and shaken at 250 rpm in a New Brunswick gyrorotatory shaker. After 8 hr at 35°, the incubation temperature is reduced to 25° and shaking is continued for an additional 16 hr before the cell-free supernatant is recovered by centrifugation. At this stage the cells should be in stationary phase of growth. Growth Conditions for the Isolation of Inactive Protoxin. The same medium is used as for mature toxin isolation except that the RNA is omitted because it has no effect on the yield of the protoxin. The flasks are inoculated as above and shaken at 35° until the optical density of the culture at 660 nm is between 8 and 10. This is the point at which the cells begin to leave log phase. It usually takes about 8 hr under these conditions. Timing of bacterial growth is critical. Very little protein is released before an OD660 of 8 is attained, and after the cells have passed an OD660 of 10, so much protease has been released that it is impossible to prevent conversion of the proaerolysin to the mature toxin during isolation. Purification of the Mature Toxin. Each stage in the purification is monitored using the titer assay. Under the conditions described here, aerolysin is the only hemolytic protein which is detected. Ammonium sulfate is added to the culture supernatant to 60% of saturation at 0°. After 4 hr the precipitate is collected by centrifugation at 10,000 g for 30 min and extracted twice with 0.03 M sodium borate, pH 8.2 (10 ml of this buffer is used for every 1 liter of the original culture supernatant). Nearly all of the hemolytic activity is recovered in the supernatant of the second wash. This is desalted by passing it down a 2.5 x 25 cm column of Sephadex G-25 equilibrated in the 0.03 M sodium borate buffer, pH 8.2. It is essential that this and all subsequent steps be performed at 0° as concentrated solutions of the active toxin aggregate spontaneously and irreversibly upon warming. The void volume fraction from the Sephadex G-25 column is applied to a 1.5 x 16 cm column of DEAE-Sepharose CL-6B also equilibrated in the 0.03 M borate buffer. The column is eluted at 15 ml/hr with a linear gradient obtained by mixing 250 ml of the 0.03 M borate with 0.3 M NaCI in same buffer. Four-milliliter fractions are collected. The hemolytic protein, which is eluted with 0.12 M NaC1, is precipitated by adding amino8 j. T. Buckley, L. N. Halasa, K. D. Lund, and S. Maclntyre, Can. J. Biochem. 59, 430 (1981).
196
PREPARATION OF TOXINS
[27]
nium sulfate to 60% of saturation at 0°. The precipitate is dissolved in a small amount of 0.03 M borate, pH 8.2, and applied to a 1.5 × 60 cm Sephadex G-150 column. Active fractions, eluted from this column at 12 ml/hr in the same buffer, are combined and applied to a hydroxyapatite column equilibrated in 0.01 M phosphate, pH 7. This column is eluted at 15 ml/hr using a linear gradient of 250 ml 0.01 M phosphate and 250 ml of 0.25 M phosphate, pH 7. The hydroxyapatite is obtained from Bio-Rad and prepared according to the company's instructions. Pure aerolysin is recovered from the column in fractions which are approximately 0.05 M phosphate. The results of a representative purification are presented in Table I. Purification of the Protoxin. 3 Ammonium sulfate is added to culture supernatants to 85% of saturation of 0° and the mixture is allowed to stand for at least 4 hr. The precipitate is collected by centrifugation at 10,000 g for 30 min and resuspended in 20 mM phosphate-0.3 M NaC1, pH 6 (10 ml/liter of culture supernatant). At this stage the protoxin is quite stable as long as the suspension is not warmed. During purification, stability is higher at pH values below 7 and in solutions of high ionic strength, especially solutions containing divalent anions such as sulfate and phosphate. All subsequent steps should be carried out without delay. The suspension is centrifuged (15,000 g, 30 min) and the clear supernatant is desalted on a 2.5 × 30 cm column of Sephadex G-25 equilibrated in the same buffer. Void volume fractions are applied directly to a 1.6 × 15 cm column of hydroxyapatite (see above) also equilibrated in the NaCl-phosphate buffer. This is eluted at 20 ml/hr with a linear gradient formed by mixing 250 ml of the starting buffer and 250 ml of 0.15 M phosphate-0.3 M NaCl (pH 6). Fractions in a broad, sometimes double peak, eluting at -0.05 M phosphate, contain protoxin and varying amounts of the mature TABLE I PURIFICATION OF AEROLYSIN a
Step
Total protein (mg)
Culture s u p e r n a t a n t b Second w a s h DEAE S e p h a d e x G-150 Hydroxyapatite
657 189 17 7.5 4.5
Total H U 5.66 3.09 3.63 1.76 1.22
× x x × x
106 106 105 105 105
Activity (%) 100 55 6.4 3.1 2.2'
a F r o m B u c k l e y e t al. s b An aliquot o f 1200 ml o f culture s u p e r n a t a n t was used.
Specific activity ( H U - mg protein -~) 0.86 1.66 2.14 2.46 2.68
× × × × ×
104 104 104 104 104
[27]
AEROLYSINFROMA. hydrophila
197
toxin (Fig. 1A). At this and all other stages, the relative amounts of toxin and protoxin can be estimated by determining the degree of activation obtained with trypsin, or by SDS-PAGE. 9 Fractions from the first half of the double peak obtained from the hydroxyapatite column are enriched in the protoxin (Fig. 1B). These are combined and the protein precipitated by adding ammonium sulfate to 85% of saturation. The precipitate is recovered by centrifugation at 15,000 g for 30 min and dissolved in 20 mM Tris-5 mM EDTA-0.5 mM phenylmethylsulfonyl fluoride, pH 8. The protein is then applied to a DEAE-Sepharose CL-6B column equilibrated in the same buffer and eluted at 20 ml/hr with a linear gradient of 250 ml of 20 mM Tris and 250 ml of 20 mM Tris-0.5 M NaC1, pH 8. The protoxin elutes immediately after the mature protein. Properties Purity and Molecular Weight. Aerolysin is isolated free of measurable proteases by these procedures and migrates as a single band on SDSPAGE, accounting for at least 95% of the applied protein. In nondenaturing acrylamide gels, 1° the toxin separates into two bands of approximately equal intensities, both of which are hemolytic. This is consistent with the observation that two aerolysin isomers are isolated together, with isoelectric points of 5.39 and 5.46. 8 Our preliminary evidence suggests that the existence of isomers is due to posttranslational modification of the protein (unpublished observations). Proaerolysin samples often appear free of contaminating aerolysin as well as other proteins by SDS-PAGE, but they always have some residual hemolytic activity. This may be due to traces of contaminating toxin, although it is possible that the protoxin is weakly active. The apparent molecular weight of the mature toxin, determined by SDS-PAGE, is about 51,500. A similar value is obtained by gel filtration on Sephadex G150. The molecular weight of the protoxin, also determined by SDSPAGE, is 54,000. Activation of the Protoxin. 3 Proaerolysin is completely converted to the mature active protein by treatment with 0.1-1.0 /zg/ml of trypsin (ratio of protease to protoxin 1 : 100) for as little as 5 rain. Conversion is due to the removal of - 2 0 amino acids from the C-terminus of the protoxin. The mature toxin is not further degraded by trypsin even when the concentration of the protease is increased 10-fold and the incubation is continued for 1 hr. The protoxin can also be activated by treatment with 9 D. M. Neville, J. Biol. Chem. 246, 6328 (1971). io B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).
198
PREPARATION OF TOXINS
[27]
A
!, O
E 9" O o0 o,I
o
fo'\
£
/\
O ,.Q
,<
°'°~
n
I 20
o o • o %,o" o.O,o.o.O,o.O, o.
I 40
°.o
.O.o.o.o. °
I 60
Fraction
B
FIG. 1. Hydroxyapatite chromatography of toxin and protoxin. (A) Elution profile from a hydroxyapatite column (1.6 by 15 cm). Desalted ammonium sulfate precipitate (20 ml) was applied and eluted as described in the text. Only half of the profile is shown; the other half contained no material with an absorbance at 280 nm. The arrow marks the fraction with the highest hemolytic titer. (B) SDS-PAGE of column fractions. The labels refer to fraction numbers. The unlabeled lane contains purified mature aerolysin. Reproduced from Howard and Buckley. 3
[27]
AEROLYSlN FROMA. hydrophila
199
T A B L E II AMINO ACID COMPOSITION OF MATURE AEROLYSIN a
Amino acid
Number of residues b
Amino acid
Number of residues b
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine
22 7 16 64 31 38 37 24 49
Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
28 ND c 37 3 17 25 24 14 ND
a Determined as described in J. T. Buckley, L. N. Halasa, and S. MacIntyre, J. Biol. Chem. 257, 3320 (1982). b Moles of amino acid per mole of enzyme. N D , not determined.
similar amounts of thermolysin and chymotrypsin as well as with an Aeromonas protease, but not by staphylococcal V8 protease, which destroys both forms of the toxin. Stability. Like many other hole-forming toxins, aerolysin aggregates spontaneously in concentrated solutions. The process is greatly accelerated at temperatures above 0 °. Aggregation also occurs during freezing and thawing. The process is essentially irreversible (aggregates are not dissociated by boiling in S D S - P A G E sample buffer, although they are disrupted by 6 M urea) and results in loss of activity. Because aggregated protein may interfere in some experiments, samples of the mature protein are routinely centrifuged (20,000 g, 30 min) immediately before use. In contrast to aerolysin, proaerolysin, once purified and freed of protease, is stable at temperatures above 0 ° and to repeated freezing and thawing. Preliminary experiments indicate that the protoxin is incapable of forming aggregates and that this may be the reason for its very low holeforming activity. Amino Acid Composition and Sequence of the Amino Terminus. The amino acid composition of aerolysin is presented in Table II. It is very similar to the amino acid composition predicted by the nucleotide sequence of the gene (unpublished observations). The amino-terminal amino acid is alanine. 3 The amino acid sequence at the amino terminus, translated from the D N A sequence, confirms the published amino acid sequence at this end of aerolysin, except at position 19, where serine was reported and the nucleotide sequence is translated as cysteine.
[27]
193
examination shows erosive lesions and desquamation of necrotic mucosa accompanied by marked neutrophil infiltration into the intestinal wall. Low doses of the thermostable direct hemolysin rapidly kill various experimental animals, such as mice, rats, and guinea pigs. For example, intravenous injection of 5/xg of the hemolysin killed mice within 1 min. The rapid death of the animals was shown to be due to the cardiotoxicity of the thermostable direct hemolysin. 4,1° ~0 K. Goshima, T. Honda, M. Hirata, K. Kikuchi, Y. Takeda, and T. Miwatani, J. Mol. Cell. Cardiol. 9, 191 (1977).
[27] A e r o l y s i n f r o m A e r o m o n a s hydrophila
By J. THOMAS BVCKLEY and S. PETER HOWARD Aerolysin is a soluble cytolytic protein produced by the gram-negative bacteria Aeromonas hydrophila.l,2 It is released to the extracellular medium as an inactive protoxin (proaerolysin) which is converted to the active protein by one or more extracellular proteases which are also exported by A. hydrophila. 3 The mechanism of action of the toxin is very similar to the mechanism proposed for staphylococcal alpha toxin. 4 The toxin binds to a specific glycoprotein receptor on the surface of eukaryotic cells, inserts into the lipid bilayer, and forms holes approximately 3 nm in diameter. This results in destruction of the membrane permeability barrier and cell death? Assay Method
Principle. By far the easiest methods of measuring the activity of aerolysin are based on the ability of the toxin to cause the hemolysis of red cells. Plate assays measure the clearing of red cell suspensions in wells and the absence of cell pellets at the bottom of wells. For convenience, human red cells obtained from outdated blood are normally used; F. H. Castelitz and R. Gunther, Zentralbl. Bakteriol., Parasitenk. Infektionskr. Hyg. Abt. 1: Orig. 180, 30 (1960). B. Wretlind, R. MOllby, and T. WadstrOm, Infect. lmrnun. 4, 503 (1971). 3 S. P. Howard and J. T. Buckley, J. Bacteriol. 163, 336 (1985). 4 S. Harshman, Mol. Cell. Biochem. 23, 143 (1979). 5 S. P. Howard and J. T. Buckley, Biochemistry 21, 1662 (1982).
METHODS IN ENZYMOLOGY, VOL. 165
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
194
PREPARATION OF TOXINS
[27]
however, sensitivity can be increased more than 100-fold by using rat erythrocytes.6
Reagents PBS-albumin: 10 mM taining 0.1% bovine Washed erythrocytes: man blood, washed PBS-albumin
sodium phosphate, 150 mM NaC1, pH 7.4, conserum albumin Packed human erythrocytes from outdated hu3 × with PBS and resuspended to 0.8% (v/v) in
Assay of the Active Toxin. Hemolytic activity determinations are carried out in microtiter plates after 2-fold serial dilutions of samples in 0.1 ml of PBS-albumin. An equal volume of the 0.8% erythrocytes in PBSalbumin is added to each well and the plates are incubated at 37° for 1 hr. Hemolytic activity is expressed as the inverse of the largest dilution at which complete lysis occurs. Assay of the Inactive Protoxin. The assay procedure is essentially the same as the procedure for the mature protein. The protoxin is activated in the first well of the titer plate, which for this purpose contains PBS but no albumin. Activation is achieved by adding trypsin to a final concentration of 0.1-1 /xg/ml and incubating at room temperature for 5 min. Serial dilutions into PBS-albumin are performed and the assay continued as above. The presence of trypsin inhibitor at a final concentration of 1 mg/ml in the PBS-albumin used for serial dilution has no affect on the results of the assay. Enzyme Production and Isolation
Medium. Aeromonas hydrophila is grown in 2-liter flasks, each containing 200 ml of medium prepared by dissolving 8 g of yeast extract (Difco) and 4 g of casamino acids (Difco) in a total of 156 ml of water containing 0.2 ml of 3.3 x 10-3% thiamin and 0.2 ml of 0.12% nicotinic acid stock and adjusting the pH to 7.2. Yeast RNA (20 ml of a 10% solution, pH 7.2) is also added to each flask followed by 2 ml of 0.1 M MgSO4, 2 ml of 0.01 M CaCI2, and 20 ml 10× M9 salts (60.34 g Na2HPO4, I0.19 g NH4C1, 30.4 g KH2PO4, and 5.19 g of NaCI dissolved in water to 1 liter). The hemolytic activity of extracellular culture supernatants is far higher when RNA is present in the culture medium. 7 The reason for this has not been established, but may be due to the ability of the RNA to 6 A. W. Bernheimer, L. S. Avigad, and G. Avigad, Infect. Immun. 11, 1312 (1975). 7 A. W. Bernheimer and L. S. Avigad, Infect. Imrnun. 9, 1016 (1974).
[27]
AEROLYSINFROMA. hydrophila
195
complex divalent cations and thereby inhibit one or more extracellular proteases. Growth Conditions for the Isolation of Mature Active Toxin. 8 The flasks are inoculated with 1% of an overnight culture containing 2 × 10 9 to 5 X 10 9 viable cells per milliliter and shaken at 250 rpm in a New Brunswick gyrorotatory shaker. After 8 hr at 35°, the incubation temperature is reduced to 25° and shaking is continued for an additional 16 hr before the cell-free supernatant is recovered by centrifugation. At this stage the cells should be in stationary phase of growth. Growth Conditions for the Isolation of Inactive Protoxin. The same medium is used as for mature toxin isolation except that the RNA is omitted because it has no effect on the yield of the protoxin. The flasks are inoculated as above and shaken at 35° until the optical density of the culture at 660 nm is between 8 and 10. This is the point at which the cells begin to leave log phase. It usually takes about 8 hr under these conditions. Timing of bacterial growth is critical. Very little protein is released before an OD660 of 8 is attained, and after the cells have passed an OD660 of 10, so much protease has been released that it is impossible to prevent conversion of the proaerolysin to the mature toxin during isolation. Purification of the Mature Toxin. Each stage in the purification is monitored using the titer assay. Under the conditions described here, aerolysin is the only hemolytic protein which is detected. Ammonium sulfate is added to the culture supernatant to 60% of saturation at 0°. After 4 hr the precipitate is collected by centrifugation at 10,000 g for 30 min and extracted twice with 0.03 M sodium borate, pH 8.2 (10 ml of this buffer is used for every 1 liter of the original culture supernatant). Nearly all of the hemolytic activity is recovered in the supernatant of the second wash. This is desalted by passing it down a 2.5 x 25 cm column of Sephadex G-25 equilibrated in the 0.03 M sodium borate buffer, pH 8.2. It is essential that this and all subsequent steps be performed at 0° as concentrated solutions of the active toxin aggregate spontaneously and irreversibly upon warming. The void volume fraction from the Sephadex G-25 column is applied to a 1.5 x 16 cm column of DEAE-Sepharose CL-6B also equilibrated in the 0.03 M borate buffer. The column is eluted at 15 ml/hr with a linear gradient obtained by mixing 250 ml of the 0.03 M borate with 0.3 M NaCI in same buffer. Four-milliliter fractions are collected. The hemolytic protein, which is eluted with 0.12 M NaC1, is precipitated by adding amino8 j. T. Buckley, L. N. Halasa, K. D. Lund, and S. Maclntyre, Can. J. Biochem. 59, 430 (1981).
196
PREPARATION OF TOXINS
[27]
nium sulfate to 60% of saturation at 0°. The precipitate is dissolved in a small amount of 0.03 M borate, pH 8.2, and applied to a 1.5 × 60 cm Sephadex G-150 column. Active fractions, eluted from this column at 12 ml/hr in the same buffer, are combined and applied to a hydroxyapatite column equilibrated in 0.01 M phosphate, pH 7. This column is eluted at 15 ml/hr using a linear gradient of 250 ml 0.01 M phosphate and 250 ml of 0.25 M phosphate, pH 7. The hydroxyapatite is obtained from Bio-Rad and prepared according to the company's instructions. Pure aerolysin is recovered from the column in fractions which are approximately 0.05 M phosphate. The results of a representative purification are presented in Table I. Purification of the Protoxin. 3 Ammonium sulfate is added to culture supernatants to 85% of saturation of 0° and the mixture is allowed to stand for at least 4 hr. The precipitate is collected by centrifugation at 10,000 g for 30 min and resuspended in 20 mM phosphate-0.3 M NaC1, pH 6 (10 ml/liter of culture supernatant). At this stage the protoxin is quite stable as long as the suspension is not warmed. During purification, stability is higher at pH values below 7 and in solutions of high ionic strength, especially solutions containing divalent anions such as sulfate and phosphate. All subsequent steps should be carried out without delay. The suspension is centrifuged (15,000 g, 30 min) and the clear supernatant is desalted on a 2.5 × 30 cm column of Sephadex G-25 equilibrated in the same buffer. Void volume fractions are applied directly to a 1.6 × 15 cm column of hydroxyapatite (see above) also equilibrated in the NaCl-phosphate buffer. This is eluted at 20 ml/hr with a linear gradient formed by mixing 250 ml of the starting buffer and 250 ml of 0.15 M phosphate-0.3 M NaCl (pH 6). Fractions in a broad, sometimes double peak, eluting at -0.05 M phosphate, contain protoxin and varying amounts of the mature TABLE I PURIFICATION OF AEROLYSIN a
Step
Total protein (mg)
Culture s u p e r n a t a n t b Second w a s h DEAE S e p h a d e x G-150 Hydroxyapatite
657 189 17 7.5 4.5
Total H U 5.66 3.09 3.63 1.76 1.22
× x x × x
106 106 105 105 105
Activity (%) 100 55 6.4 3.1 2.2'
a F r o m B u c k l e y e t al. s b An aliquot o f 1200 ml o f culture s u p e r n a t a n t was used.
Specific activity ( H U - mg protein -~) 0.86 1.66 2.14 2.46 2.68
× × × × ×
104 104 104 104 104
[27]
AEROLYSINFROMA. hydrophila
197
toxin (Fig. 1A). At this and all other stages, the relative amounts of toxin and protoxin can be estimated by determining the degree of activation obtained with trypsin, or by SDS-PAGE. 9 Fractions from the first half of the double peak obtained from the hydroxyapatite column are enriched in the protoxin (Fig. 1B). These are combined and the protein precipitated by adding ammonium sulfate to 85% of saturation. The precipitate is recovered by centrifugation at 15,000 g for 30 min and dissolved in 20 mM Tris-5 mM EDTA-0.5 mM phenylmethylsulfonyl fluoride, pH 8. The protein is then applied to a DEAE-Sepharose CL-6B column equilibrated in the same buffer and eluted at 20 ml/hr with a linear gradient of 250 ml of 20 mM Tris and 250 ml of 20 mM Tris-0.5 M NaC1, pH 8. The protoxin elutes immediately after the mature protein. Properties Purity and Molecular Weight. Aerolysin is isolated free of measurable proteases by these procedures and migrates as a single band on SDSPAGE, accounting for at least 95% of the applied protein. In nondenaturing acrylamide gels, 1° the toxin separates into two bands of approximately equal intensities, both of which are hemolytic. This is consistent with the observation that two aerolysin isomers are isolated together, with isoelectric points of 5.39 and 5.46. 8 Our preliminary evidence suggests that the existence of isomers is due to posttranslational modification of the protein (unpublished observations). Proaerolysin samples often appear free of contaminating aerolysin as well as other proteins by SDS-PAGE, but they always have some residual hemolytic activity. This may be due to traces of contaminating toxin, although it is possible that the protoxin is weakly active. The apparent molecular weight of the mature toxin, determined by SDS-PAGE, is about 51,500. A similar value is obtained by gel filtration on Sephadex G150. The molecular weight of the protoxin, also determined by SDSPAGE, is 54,000. Activation of the Protoxin. 3 Proaerolysin is completely converted to the mature active protein by treatment with 0.1-1.0 /zg/ml of trypsin (ratio of protease to protoxin 1 : 100) for as little as 5 rain. Conversion is due to the removal of - 2 0 amino acids from the C-terminus of the protoxin. The mature toxin is not further degraded by trypsin even when the concentration of the protease is increased 10-fold and the incubation is continued for 1 hr. The protoxin can also be activated by treatment with 9 D. M. Neville, J. Biol. Chem. 246, 6328 (1971). io B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).
198
PREPARATION OF TOXINS
[27]
A
!, O
E 9" O o0 o,I
o
fo'\
£
/\
O ,.Q
,<
°'°~
n
I 20
o o • o %,o" o.O,o.o.O,o.O, o.
I 40
°.o
.O.o.o.o. °
I 60
Fraction
B
FIG. 1. Hydroxyapatite chromatography of toxin and protoxin. (A) Elution profile from a hydroxyapatite column (1.6 by 15 cm). Desalted ammonium sulfate precipitate (20 ml) was applied and eluted as described in the text. Only half of the profile is shown; the other half contained no material with an absorbance at 280 nm. The arrow marks the fraction with the highest hemolytic titer. (B) SDS-PAGE of column fractions. The labels refer to fraction numbers. The unlabeled lane contains purified mature aerolysin. Reproduced from Howard and Buckley. 3
[27]
AEROLYSlN FROMA. hydrophila
199
T A B L E II AMINO ACID COMPOSITION OF MATURE AEROLYSIN a
Amino acid
Number of residues b
Amino acid
Number of residues b
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine
22 7 16 64 31 38 37 24 49
Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
28 ND c 37 3 17 25 24 14 ND
a Determined as described in J. T. Buckley, L. N. Halasa, and S. MacIntyre, J. Biol. Chem. 257, 3320 (1982). b Moles of amino acid per mole of enzyme. N D , not determined.
similar amounts of thermolysin and chymotrypsin as well as with an Aeromonas protease, but not by staphylococcal V8 protease, which destroys both forms of the toxin. Stability. Like many other hole-forming toxins, aerolysin aggregates spontaneously in concentrated solutions. The process is greatly accelerated at temperatures above 0 °. Aggregation also occurs during freezing and thawing. The process is essentially irreversible (aggregates are not dissociated by boiling in S D S - P A G E sample buffer, although they are disrupted by 6 M urea) and results in loss of activity. Because aggregated protein may interfere in some experiments, samples of the mature protein are routinely centrifuged (20,000 g, 30 min) immediately before use. In contrast to aerolysin, proaerolysin, once purified and freed of protease, is stable at temperatures above 0 ° and to repeated freezing and thawing. Preliminary experiments indicate that the protoxin is incapable of forming aggregates and that this may be the reason for its very low holeforming activity. Amino Acid Composition and Sequence of the Amino Terminus. The amino acid composition of aerolysin is presented in Table II. It is very similar to the amino acid composition predicted by the nucleotide sequence of the gene (unpublished observations). The amino-terminal amino acid is alanine. 3 The amino acid sequence at the amino terminus, translated from the D N A sequence, confirms the published amino acid sequence at this end of aerolysin, except at position 19, where serine was reported and the nucleotide sequence is translated as cysteine.