Vol. 21, No. 1
INFECTION AND IMMUNITY, July 1978, p. 124-128 0019-9567/78/0021-0124$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Further Characterization of Mycobacterium ulcerans Toxin WAYNE T. HOCKMEYER,' RICHARD E. KRIEG,* M. REICH,2 AND ROY D. JOHNSON' Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306,' and the Department of Microbiology, The George Washington University School of Medicine, Washington, D.C. 200372 Received for publication 16 January 1978
Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chloroform-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a highdensity lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex.
Mycobacterium ulcerans produces an exoIsolation of toxin. When an aliquot of the culture toxin in culture which, when inoculated into filtrate (CF) produced a 90% cytopathic effect (CPE)
guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes which resemble infections of humans (6, 9). Preliminary reports indicate that this exotoxin is heat stable and ether insoluble and that it is a protein of low molecular weight. Herein, we further describe some additional biochemical and physiochemical properties of this toxin. MATERIALS AND METHODS Organisms. Cultures of M. ulcerans were obtained from Wayne M. Meyers, who isolated them from patients with typical lesions in the Republic of Zaire (8). M. ulcerans was cultured and grown as previously described (6). Briefly, this involved isolation of the organism from ulcers of patients with active infection, decontamination with oxalic acid, and inoculation onto Lowenstein-Jensen medium. Purity of the isolates was determined by streaking Middlebrook agar plates and by microscopic examination of Ziehl-Neelsen stains. The organisms were grown for 6 weeks at 320C in Dubos broth base supplemented with 5% bovine serum albumin (fraction V) and 7.5% dextrose. Roller bottles containing 500 ml of Dubos broth were inoculated with M. ulcerans and incubated to a final concentration of approximately 109 cells per ml. To prevent loss of toxicity, the cultures were periodically passed through mouse footpads, isolated, and inoculated onto Lowenstein-Jensen slants; the isolated colonies were then reinoculated into Dubos broth.
on monolayer cultures of L929 cells, the cultures were centrifuged at 12,062 x g for 15 min and the CF was concentrated to 40 mg of protein per ml with an Amicon ultrafiltration system having a UM 05 filter. The concentrated CF was sterilized by filtration through a 0.22-jIm filter. The sterility of the CF was tested by plating on Middlebrook agar and blood agar
and by examination of Ziehl-Neelsen- and Gramstained smears. This material served as one source of toxin. Isolation of lipoproteins. The lipoproteins present in the CF were isolated by KBr isopycnic density gradient ultracentrifugation (5). The CF was layered on a solution of KBr and centrifuged at 14,,000 x g for 48 h in a Beckman SW41 rotor. The final density of the solution to be centrifuged, after addition of the sample, was 1.20 g/cm3 as determined by refractometry. After 48 h of centrifugation, the opaque layer at the top of the tube was carefully removed with a Pasteur pipette. The yellowish protein-rich layer on the bottom of the tube was also isolated. The upper high-density lipoprotein (HDL) layer and the lower protein-rich layer were exhaustively dialyzed against 0.01 M phosphate-buffered saline (PBS), pH 7.2, at 4°C and then concentrated by ultrafiltration with a UM 05 membrane. The HDL and lower protein-rich samples were then tested for toxicity. Assay for exotoxin activity. The toxicity of various preparations was determined by inoculating monolayer cultures of L929 cells as previously described (6). The percentage of cells affected (detached, ovoid, enucleated) was measured and expressed as percent 124
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CPE when compared to controls after 48 h of incubation at 370C. Effect of heat. Ten-milliliter samples of CF (9 mg of protein per ml) and HDL toxin (1.2 mg of protein per ml) were heated at 60, 70, 80, 90, and 1000C and autoclaved at 1210C for 30 min. After heat treatment, the samples were then centrifuged at 4,300 x g for 20 min. The supernatant was removed with a Pasteur pipette, and the precipitate was resuspended in 10 ml of 0.015 M PBS (pH 7.2) for toxicity tests. Untreated CF and PBS served as positive and negative controls, respectively. Effect of pH. Two-milliliter samples of CF (9 mg of protein per ml) were exposed to acid or alkali for 4 h at 370C by titrating the samples to pH 1, 3, 5, 7, 8, 9, or 11 with 1 N NaOH or 1 N HCl. After incubation, the samples were readjusted to pH 7.0. Two-milliter samples of 0.015 M PBS (pH 7.2), which were titrated to the appropriate pH and incubated and retitrated to neutrality, and untreated CF served as controls. Effect of proteolytic enzymes. CF was dialyzed overnight at 40C against 0.05 M PBS, pH 7.4. Pronase (Sigma Chemical Co., St. Louis, Mo.) was added directly to the toxin to yield final protein ratios of toxin to enzyme of 50:1, 25:1, 20:1, 10:1, and 5:1. The mixture was incubated for 24 h at 370C. The samples were then redialyzed overnight at 40C against 0.05 M PBS, pH 7.4. If necessary, the samples were brought back to the original protein concentration by pressure dialysis with an Amicon UM 05 filter. The samples were sterilized by filtration through 0.22-gm filters and tested for toxicity. Trypsin (Sigma) was dissolved in 0.2 ml of 0.005 M HCI and added to samples of CF to achieve final protein ratios of toxin to enzyme of 20:1, 10:1, and 5:1. The samples were treated and tested as described above for the Pronase experiments. Untreated CF or PBS with trypsin or Pronase at the desired concentrations served as controls. Effect of other various hydrolytic enzymes. Aliquots of CF were dialyzed against each of the buffers used to prepare the hydrolytic enzymes to ensure optimal conditions for enzyme activity. One milliliter of the dialyzed CF (9 mg of protein per ml) was mixed with 1 ml of enzyme (1 mg/ml unless otherwise noted), incubated at 370C for 2 h, dialyzed against 0.1 M PBS (pH 7.2), passed through a 0.22-ILm filter, and tested for toxicity. The following enzymes were used: phospholipases A, C, and D (Sigma), 0.2, 0.08, and 1.0 mg/ml, respectively, in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.0) containing 0.6% CaCl2; lipase (Sigma; and Worthington Biochemicals Corp., Freehold, N.J.) in 0.05 M Tris-hydrochloride (pH 8.0) containing 0.06% CaCl2; dextranase (Worthington) in 0.1 M phosphate buffer, pH 6.0; cellulose (Sigma) in 0.05 M acetate buffer, pH 5.0; a-amylase (Sigma) in 0.1 M phosphate buffer (pH 7.0) containing 0.01 M NaCl .-amylase (Worthington) in 0.02 M acetate, pH 5.0; a-glucosidase (Sigma), 0.7 mg/mil in 0.1 M phosphate buffer, pH 6.0; f6-glucosidase (Sigma) in 0.1 M acetate buffer, pH 5.0, lysozyme (Sigma) in 0.1 M glycine-NaOH buffer, pH 9.0; hyaluronidase (Sigma) in 0.1 M acetate buffer (pH 5.0) containing 0.15 M NaCl; neuraminidase (Sigma) in 0.1 M acetate buffer,
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pH 5.0, collagenase (Sigma) in 0.1 M Tris buffer (pH 7) containing 0.004% CaC12. Untreated CF mixed with the appropriate buffers and buffers containing enzymes served as controls. Effects of various chemical reagents. Ammonium sulfate was added to 1 liter of CF (9 mg of protein per ml) in increasing amounts to final concentrations up to 65% saturation. The solution was observed for a visible precipitate and then centrifuged at 4,400 x g for 30 min. Any precipitate was resuspended in 0.01 M PBS (pH 7.4) and dialyzed against 0.01 M phosphate buffer (pH 7.4) at 40C before testing for toxicity. Samples of CF (2.5, 5, and 7.5 mg of protein per ml) were treated with 2-mercaptoethanol at concentrations from 0.02 to 0.2 M and alkylated as per procedures used for immunoglobulins (3). After reduction and alkylation, the samples were dialyzed against repeated changes of 0.01 M PBS (pH 7.2) at 40C. The samples were sterilized with a 0.22jpm filter and then tested for toxicity. CF (12 mg of protein per ml) was treated with a solution of 4.0 M urea and 0.05 M sodium acetate (11). After incubation at 300C for 2 h, the mixture was exhaustively dialyzed against 1 liter of 0.05 M sodium phosphate buffer, pH 7.2. After dialysis, aliquots of the urea-treated material were tested for toxicity. CF was also treated with 6 M guanidine hydrochloride and tested for toxicity in the same manner as described for urea. Samples of CF (5 mg of protein per ml) were treated with sodium m-periodate according to procedures used for glycopeptides or glycoproteins (10). Oxidation was carried out in 0.05 M sodium acetate buffer (pH 4.5) at 40C in the dark. The samples were exposed to concentrations of sodium m-periodate varying from 0.0025 to 0.08 M at 15, 30, and 48 h and then tested for toxicity. One-milliliter samples of CF were dialyzed against 0.1 M phosphate buffer (pH 7.0) at 40C and mixed with 1 ml of solutions (2 mg/ml) of p-chloromercuribenzoate, ethylenediaminetetraacetate, sodium dodecyl sulfate, and sodium deoxycholate, all of which were dissolved in 0.1 M phosphate buffer, pH 7.0. The preparations were incubated at room temperature for 2 h, dialyzed against 0.1 M phosphate buffer (pH 7.2), passed through a 0.22-gtm filter, and stored at 40C until tested for toxicity. Controls for each of the above experiments were untreated aliquots of original CF and buffers containing the chemical agents. Ultrafiltration. A 500-ml volume of CF was diluted with 500 ml of distilled water and placed in an ultrafiltration system (Amicon) fitted with an XM 300 membrane. Ultrafiltration continued until 50 ml of fluid remained above the membrane. This material (XM 300 retentate) was mixed with 5 volumes of PBS (pH 7.2), reconcentrated to 50 ml, filter sterilized, and preserved at 40C for toxicity tests. The XM 300 dialysate, including washings, was concentrated to 500 ml by a PM 10 membrane, and an aliquot was saved for toxicity testing. Sequential ultrafiltration was continued as described above with XM 100, XM 50, and PM 30 membranes. Organic solvent extraction. A 500-ml amount of
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CF was rotary evaporated to dryness, and the residue was extracted with chloroform-methanol (2) and with ether. The chloroform-methanol fraction was vacuum dried, and the organic-soluble residue was resuspended in 0.01 M PBS, pH 7.2. The organic-insoluble material was also resuspended in 0.01 M PBS (pH 7.2), and both fractions were tested for toxicity. Rotary evaporated CF was extracted with ether by adding 50 ml to the rotary flask and mixing for 30 min. The ether fraction was removed, and an additional 50 ml of ether was added. The two ether fractions were combined and vacuum dried, and the residue was suspended in 0.01 M PBS, pH 7.2. The organic-insoluble material was also suspended in 0.01 M PBS (pH 7.2), and both fractions were tested for toxicity.
Chemical analysis. Protein was analyzed by the method of Lowry et al. with bovine serum albumin, fraction V (Miles Laboratory, Elkhart, Ind.), as the standard (7). Carbohydrate was assayed by the phenol-sulfuric acid method with glucose, galactose, maltose, and xylose as standards (1).
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RESULTS M. ulcerans CF and HDL toxins are heat resistant as shown in Fig. 1, the heat inactivation curve. CF begins to form a noticeable precipitate at 80, 90, and 1000C, with a corresponding loss of 25% of its cytopathic activity. The precipitate itself, however, is not inactivated at these temperatures, since it retains a 75% CPE when resuspended in PBS and tested in L929 cells. It is probable that the toxin is coprecipitating with other proteins in the heated CF but remains active. This is supported by the observation that the more purified HDL toxin shows no loss of CPE through the entire temperature range to 1000C and forms very little visible precipitate. Whatever precipitate is formed is not toxic. Both the CF and HDL toxins are heat inactivated by autoclaving at 1210C.
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INFECTION AND IMMUNITY, July 1978, p. 124-128 0019-9567/78/0021-0124$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Further Characterization of Mycobacterium ulcerans Toxin WAYNE T. HOCKMEYER,' RICHARD E. KRIEG,* M. REICH,2 AND ROY D. JOHNSON' Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306,' and the Department of Microbiology, The George Washington University School of Medicine, Washington, D.C. 200372 Received for publication 16 January 1978
Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chloroform-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a highdensity lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex.
Mycobacterium ulcerans produces an exoIsolation of toxin. When an aliquot of the culture toxin in culture which, when inoculated into filtrate (CF) produced a 90% cytopathic effect (CPE)
guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes which resemble infections of humans (6, 9). Preliminary reports indicate that this exotoxin is heat stable and ether insoluble and that it is a protein of low molecular weight. Herein, we further describe some additional biochemical and physiochemical properties of this toxin. MATERIALS AND METHODS Organisms. Cultures of M. ulcerans were obtained from Wayne M. Meyers, who isolated them from patients with typical lesions in the Republic of Zaire (8). M. ulcerans was cultured and grown as previously described (6). Briefly, this involved isolation of the organism from ulcers of patients with active infection, decontamination with oxalic acid, and inoculation onto Lowenstein-Jensen medium. Purity of the isolates was determined by streaking Middlebrook agar plates and by microscopic examination of Ziehl-Neelsen stains. The organisms were grown for 6 weeks at 320C in Dubos broth base supplemented with 5% bovine serum albumin (fraction V) and 7.5% dextrose. Roller bottles containing 500 ml of Dubos broth were inoculated with M. ulcerans and incubated to a final concentration of approximately 109 cells per ml. To prevent loss of toxicity, the cultures were periodically passed through mouse footpads, isolated, and inoculated onto Lowenstein-Jensen slants; the isolated colonies were then reinoculated into Dubos broth.
on monolayer cultures of L929 cells, the cultures were centrifuged at 12,062 x g for 15 min and the CF was concentrated to 40 mg of protein per ml with an Amicon ultrafiltration system having a UM 05 filter. The concentrated CF was sterilized by filtration through a 0.22-jIm filter. The sterility of the CF was tested by plating on Middlebrook agar and blood agar
and by examination of Ziehl-Neelsen- and Gramstained smears. This material served as one source of toxin. Isolation of lipoproteins. The lipoproteins present in the CF were isolated by KBr isopycnic density gradient ultracentrifugation (5). The CF was layered on a solution of KBr and centrifuged at 14,,000 x g for 48 h in a Beckman SW41 rotor. The final density of the solution to be centrifuged, after addition of the sample, was 1.20 g/cm3 as determined by refractometry. After 48 h of centrifugation, the opaque layer at the top of the tube was carefully removed with a Pasteur pipette. The yellowish protein-rich layer on the bottom of the tube was also isolated. The upper high-density lipoprotein (HDL) layer and the lower protein-rich layer were exhaustively dialyzed against 0.01 M phosphate-buffered saline (PBS), pH 7.2, at 4°C and then concentrated by ultrafiltration with a UM 05 membrane. The HDL and lower protein-rich samples were then tested for toxicity. Assay for exotoxin activity. The toxicity of various preparations was determined by inoculating monolayer cultures of L929 cells as previously described (6). The percentage of cells affected (detached, ovoid, enucleated) was measured and expressed as percent 124
VOL. 21, 1978
CPE when compared to controls after 48 h of incubation at 370C. Effect of heat. Ten-milliliter samples of CF (9 mg of protein per ml) and HDL toxin (1.2 mg of protein per ml) were heated at 60, 70, 80, 90, and 1000C and autoclaved at 1210C for 30 min. After heat treatment, the samples were then centrifuged at 4,300 x g for 20 min. The supernatant was removed with a Pasteur pipette, and the precipitate was resuspended in 10 ml of 0.015 M PBS (pH 7.2) for toxicity tests. Untreated CF and PBS served as positive and negative controls, respectively. Effect of pH. Two-milliliter samples of CF (9 mg of protein per ml) were exposed to acid or alkali for 4 h at 370C by titrating the samples to pH 1, 3, 5, 7, 8, 9, or 11 with 1 N NaOH or 1 N HCl. After incubation, the samples were readjusted to pH 7.0. Two-milliter samples of 0.015 M PBS (pH 7.2), which were titrated to the appropriate pH and incubated and retitrated to neutrality, and untreated CF served as controls. Effect of proteolytic enzymes. CF was dialyzed overnight at 40C against 0.05 M PBS, pH 7.4. Pronase (Sigma Chemical Co., St. Louis, Mo.) was added directly to the toxin to yield final protein ratios of toxin to enzyme of 50:1, 25:1, 20:1, 10:1, and 5:1. The mixture was incubated for 24 h at 370C. The samples were then redialyzed overnight at 40C against 0.05 M PBS, pH 7.4. If necessary, the samples were brought back to the original protein concentration by pressure dialysis with an Amicon UM 05 filter. The samples were sterilized by filtration through 0.22-gm filters and tested for toxicity. Trypsin (Sigma) was dissolved in 0.2 ml of 0.005 M HCI and added to samples of CF to achieve final protein ratios of toxin to enzyme of 20:1, 10:1, and 5:1. The samples were treated and tested as described above for the Pronase experiments. Untreated CF or PBS with trypsin or Pronase at the desired concentrations served as controls. Effect of other various hydrolytic enzymes. Aliquots of CF were dialyzed against each of the buffers used to prepare the hydrolytic enzymes to ensure optimal conditions for enzyme activity. One milliliter of the dialyzed CF (9 mg of protein per ml) was mixed with 1 ml of enzyme (1 mg/ml unless otherwise noted), incubated at 370C for 2 h, dialyzed against 0.1 M PBS (pH 7.2), passed through a 0.22-ILm filter, and tested for toxicity. The following enzymes were used: phospholipases A, C, and D (Sigma), 0.2, 0.08, and 1.0 mg/ml, respectively, in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.0) containing 0.6% CaCl2; lipase (Sigma; and Worthington Biochemicals Corp., Freehold, N.J.) in 0.05 M Tris-hydrochloride (pH 8.0) containing 0.06% CaCl2; dextranase (Worthington) in 0.1 M phosphate buffer, pH 6.0; cellulose (Sigma) in 0.05 M acetate buffer, pH 5.0; a-amylase (Sigma) in 0.1 M phosphate buffer (pH 7.0) containing 0.01 M NaCl .-amylase (Worthington) in 0.02 M acetate, pH 5.0; a-glucosidase (Sigma), 0.7 mg/mil in 0.1 M phosphate buffer, pH 6.0; f6-glucosidase (Sigma) in 0.1 M acetate buffer, pH 5.0, lysozyme (Sigma) in 0.1 M glycine-NaOH buffer, pH 9.0; hyaluronidase (Sigma) in 0.1 M acetate buffer (pH 5.0) containing 0.15 M NaCl; neuraminidase (Sigma) in 0.1 M acetate buffer,
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pH 5.0, collagenase (Sigma) in 0.1 M Tris buffer (pH 7) containing 0.004% CaC12. Untreated CF mixed with the appropriate buffers and buffers containing enzymes served as controls. Effects of various chemical reagents. Ammonium sulfate was added to 1 liter of CF (9 mg of protein per ml) in increasing amounts to final concentrations up to 65% saturation. The solution was observed for a visible precipitate and then centrifuged at 4,400 x g for 30 min. Any precipitate was resuspended in 0.01 M PBS (pH 7.4) and dialyzed against 0.01 M phosphate buffer (pH 7.4) at 40C before testing for toxicity. Samples of CF (2.5, 5, and 7.5 mg of protein per ml) were treated with 2-mercaptoethanol at concentrations from 0.02 to 0.2 M and alkylated as per procedures used for immunoglobulins (3). After reduction and alkylation, the samples were dialyzed against repeated changes of 0.01 M PBS (pH 7.2) at 40C. The samples were sterilized with a 0.22jpm filter and then tested for toxicity. CF (12 mg of protein per ml) was treated with a solution of 4.0 M urea and 0.05 M sodium acetate (11). After incubation at 300C for 2 h, the mixture was exhaustively dialyzed against 1 liter of 0.05 M sodium phosphate buffer, pH 7.2. After dialysis, aliquots of the urea-treated material were tested for toxicity. CF was also treated with 6 M guanidine hydrochloride and tested for toxicity in the same manner as described for urea. Samples of CF (5 mg of protein per ml) were treated with sodium m-periodate according to procedures used for glycopeptides or glycoproteins (10). Oxidation was carried out in 0.05 M sodium acetate buffer (pH 4.5) at 40C in the dark. The samples were exposed to concentrations of sodium m-periodate varying from 0.0025 to 0.08 M at 15, 30, and 48 h and then tested for toxicity. One-milliliter samples of CF were dialyzed against 0.1 M phosphate buffer (pH 7.0) at 40C and mixed with 1 ml of solutions (2 mg/ml) of p-chloromercuribenzoate, ethylenediaminetetraacetate, sodium dodecyl sulfate, and sodium deoxycholate, all of which were dissolved in 0.1 M phosphate buffer, pH 7.0. The preparations were incubated at room temperature for 2 h, dialyzed against 0.1 M phosphate buffer (pH 7.2), passed through a 0.22-gtm filter, and stored at 40C until tested for toxicity. Controls for each of the above experiments were untreated aliquots of original CF and buffers containing the chemical agents. Ultrafiltration. A 500-ml volume of CF was diluted with 500 ml of distilled water and placed in an ultrafiltration system (Amicon) fitted with an XM 300 membrane. Ultrafiltration continued until 50 ml of fluid remained above the membrane. This material (XM 300 retentate) was mixed with 5 volumes of PBS (pH 7.2), reconcentrated to 50 ml, filter sterilized, and preserved at 40C for toxicity tests. The XM 300 dialysate, including washings, was concentrated to 500 ml by a PM 10 membrane, and an aliquot was saved for toxicity testing. Sequential ultrafiltration was continued as described above with XM 100, XM 50, and PM 30 membranes. Organic solvent extraction. A 500-ml amount of
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CF was rotary evaporated to dryness, and the residue was extracted with chloroform-methanol (2) and with ether. The chloroform-methanol fraction was vacuum dried, and the organic-soluble residue was resuspended in 0.01 M PBS, pH 7.2. The organic-insoluble material was also resuspended in 0.01 M PBS (pH 7.2), and both fractions were tested for toxicity. Rotary evaporated CF was extracted with ether by adding 50 ml to the rotary flask and mixing for 30 min. The ether fraction was removed, and an additional 50 ml of ether was added. The two ether fractions were combined and vacuum dried, and the residue was suspended in 0.01 M PBS, pH 7.2. The organic-insoluble material was also suspended in 0.01 M PBS (pH 7.2), and both fractions were tested for toxicity.
Chemical analysis. Protein was analyzed by the method of Lowry et al. with bovine serum albumin, fraction V (Miles Laboratory, Elkhart, Ind.), as the standard (7). Carbohydrate was assayed by the phenol-sulfuric acid method with glucose, galactose, maltose, and xylose as standards (1).
100
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RESULTS M. ulcerans CF and HDL toxins are heat resistant as shown in Fig. 1, the heat inactivation curve. CF begins to form a noticeable precipitate at 80, 90, and 1000C, with a corresponding loss of 25% of its cytopathic activity. The precipitate itself, however, is not inactivated at these temperatures, since it retains a 75% CPE when resuspended in PBS and tested in L929 cells. It is probable that the toxin is coprecipitating with other proteins in the heated CF but remains active. This is supported by the observation that the more purified HDL toxin shows no loss of CPE through the entire temperature range to 1000C and forms very little visible precipitate. Whatever precipitate is formed is not toxic. Both the CF and HDL toxins are heat inactivated by autoclaving at 1210C.
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