APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1978, p. 462-464
0099-2240/78/0035-0462$02.00/0 Copyright i) 1978 American Society for Microbiology
Vol. 35,I,No. 2
Printed in U.S.A.
Observations on Toxin and Hemagglutinin Produced by Clostridium botulinum Type C KEIJI OGUMA,* AKIO NAKANE, AND HIROO IIDA Department of Bacteriology, Hokkaido University, School ofMedicine, Sapporo, Japan
Received for publication 15 September 1977
In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.
The crystalline type A toxin of Clostridium botulinum, which behaves as a simple homogeneous protein and has a molecular weight of 900,000 (19S) in acid solution, dissociates into a toxic portion with a molecular weight of 150,000 (7S) and hemagglutinin (HA) in alkaline solution (1, 6). Purified type E toxin, with a molecular weight of 350,000 (12S), also separates into toxic and nontoxic components of the same molecular size (each 7S) in alkaline solution (4). It has also been reported that type A produces two different-molecular-size toxins in the culture fluid and that only the large-size toxin shows the HA activity (2, 3). Sugii and Sakaguchi (10), Kozaki et al. (5), and Ohishi and Sakaguchi (9) purified the type A, B, and F toxins by similar procedures and concluded the following. Types A, B, E, and F commonly produce 12S toxin (10S in type F), which shows no HA activity. In addition, type A produces 16S and 19S toxins, and type B produces 16S toxin. These toxins (16S and 19S) are formed by the association of HA and 12S toxin. In alkaline solution, they separate into a 7S toxic component and other nontoxic components. Recently, type D toxin was purified from HA-positive strain CB16 and HA-negative strain 1873 (7). D-CB16 strain produced 16S and 12S toxins, and D-1873 strain produced only 12S toxin. The present studies were undertaken in an attempt to demonstrate
the existence of different-molecular-size toxins in the culture fluid of HA-positive type C. The states of existence of toxin and HA in acid and alkaline solution were also studied. HA-positive type C strain Stockholm and HAnegative type D strain 1873 were incubated at 37°C for 7 days by the cellophane tube method as described previously (8). Human erythrocytes were washed three times with 0.1 M phosphatebuffered saline at pH 6.0, and a 50% suspension was made. A duplicate suspension was made with 0.1 M phosphate-buffered saline at pH 8.0. The pH of the culture fluids incubated by the cellophane tube procedure was approximately 462
5.1, and it was adjusted to 8.0 with 1 N NaOH. These pH-adjusted and nonadjusted supernatants were each mixed with an equal volume of 50% human erythrocytes at pH 8.0 or 6.0. After incubation at 37°C for 1 h, the mixtures were centrifuged at 4,000 x g for 10 min. This adsorption treatment was performed three times, and the toxin and HA titers of the supernatant obtained each time were determined. The toxin and HA titers of the supernatants mixed with the buffer in place of the cell suspension were obtained as a control. The HA activity of CStockholm was lost by the first treatment with the cells at either pH (Table 1). On the other hand, toxigenicity decreased only after the first treatment at pH 6.0. The second and third treatments with the cells at pH 6.0, as well as at pH 8.0, showed little decrease of toxin titer. In D1873, toxigenicity was not decreased at pH 6.0; the difference between the toxin titer taken at the start and the toxin titer obtained after treatments with the cells was due to the dilution caused by mixing with the cell suspension (Table 1). Therefore, it appeared that two toxins, HA associated and HA nonassociated, exist in the CStockholm culture fluid under acid conditions and that free toxin can be found under alkaline conditions. The toxin sample of C-Stockholm prepared as a control at pH 8.0 was dialyzed against 0.1 M phosphate-buffered saline at 4°C overnight to bring the pH to 6.0. After centrifugation, the supernatant was mixed with 50% human erythrocytes at pH 6.0, and then toxin and HA titers were obtained (Table 1). Both toxin and HA titers were decreased after mixing with the cells. This implies that the combination of toxin and HA is reversible. The sedimentation pattern of C-Stockholm toxin in sucrose density gradient centrifugation also supported the above findings. When the supernatant of culture fluid was centrifuged at pH 6.0 without mixing with the cells, two toxin peaks appeared (Fig. la). After this toxin prep-
463
NOTES
VOL. 35, 1978
TABLE 1. Adsorption of toxin and hemagglutinin with human erythrocytes in acid and alkaline solutiona Strain
pH
Expt 1 C-Stockholm 6.0 6.0 6.0 6.0 8.0 8.0 8.0 8.0 D-1873 6.0 6.0
No. of adsorption Adsorption Start 1 1 3 3 1 1 3 3 Start 3 3
+ + + +
+
HA titer
256 128
0099-2240/78/0035-0462$02.00/0 Copyright i) 1978 American Society for Microbiology
Vol. 35,I,No. 2
Printed in U.S.A.
Observations on Toxin and Hemagglutinin Produced by Clostridium botulinum Type C KEIJI OGUMA,* AKIO NAKANE, AND HIROO IIDA Department of Bacteriology, Hokkaido University, School ofMedicine, Sapporo, Japan
Received for publication 15 September 1977
In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.
The crystalline type A toxin of Clostridium botulinum, which behaves as a simple homogeneous protein and has a molecular weight of 900,000 (19S) in acid solution, dissociates into a toxic portion with a molecular weight of 150,000 (7S) and hemagglutinin (HA) in alkaline solution (1, 6). Purified type E toxin, with a molecular weight of 350,000 (12S), also separates into toxic and nontoxic components of the same molecular size (each 7S) in alkaline solution (4). It has also been reported that type A produces two different-molecular-size toxins in the culture fluid and that only the large-size toxin shows the HA activity (2, 3). Sugii and Sakaguchi (10), Kozaki et al. (5), and Ohishi and Sakaguchi (9) purified the type A, B, and F toxins by similar procedures and concluded the following. Types A, B, E, and F commonly produce 12S toxin (10S in type F), which shows no HA activity. In addition, type A produces 16S and 19S toxins, and type B produces 16S toxin. These toxins (16S and 19S) are formed by the association of HA and 12S toxin. In alkaline solution, they separate into a 7S toxic component and other nontoxic components. Recently, type D toxin was purified from HA-positive strain CB16 and HA-negative strain 1873 (7). D-CB16 strain produced 16S and 12S toxins, and D-1873 strain produced only 12S toxin. The present studies were undertaken in an attempt to demonstrate
the existence of different-molecular-size toxins in the culture fluid of HA-positive type C. The states of existence of toxin and HA in acid and alkaline solution were also studied. HA-positive type C strain Stockholm and HAnegative type D strain 1873 were incubated at 37°C for 7 days by the cellophane tube method as described previously (8). Human erythrocytes were washed three times with 0.1 M phosphatebuffered saline at pH 6.0, and a 50% suspension was made. A duplicate suspension was made with 0.1 M phosphate-buffered saline at pH 8.0. The pH of the culture fluids incubated by the cellophane tube procedure was approximately 462
5.1, and it was adjusted to 8.0 with 1 N NaOH. These pH-adjusted and nonadjusted supernatants were each mixed with an equal volume of 50% human erythrocytes at pH 8.0 or 6.0. After incubation at 37°C for 1 h, the mixtures were centrifuged at 4,000 x g for 10 min. This adsorption treatment was performed three times, and the toxin and HA titers of the supernatant obtained each time were determined. The toxin and HA titers of the supernatants mixed with the buffer in place of the cell suspension were obtained as a control. The HA activity of CStockholm was lost by the first treatment with the cells at either pH (Table 1). On the other hand, toxigenicity decreased only after the first treatment at pH 6.0. The second and third treatments with the cells at pH 6.0, as well as at pH 8.0, showed little decrease of toxin titer. In D1873, toxigenicity was not decreased at pH 6.0; the difference between the toxin titer taken at the start and the toxin titer obtained after treatments with the cells was due to the dilution caused by mixing with the cell suspension (Table 1). Therefore, it appeared that two toxins, HA associated and HA nonassociated, exist in the CStockholm culture fluid under acid conditions and that free toxin can be found under alkaline conditions. The toxin sample of C-Stockholm prepared as a control at pH 8.0 was dialyzed against 0.1 M phosphate-buffered saline at 4°C overnight to bring the pH to 6.0. After centrifugation, the supernatant was mixed with 50% human erythrocytes at pH 6.0, and then toxin and HA titers were obtained (Table 1). Both toxin and HA titers were decreased after mixing with the cells. This implies that the combination of toxin and HA is reversible. The sedimentation pattern of C-Stockholm toxin in sucrose density gradient centrifugation also supported the above findings. When the supernatant of culture fluid was centrifuged at pH 6.0 without mixing with the cells, two toxin peaks appeared (Fig. la). After this toxin prep-
463
NOTES
VOL. 35, 1978
TABLE 1. Adsorption of toxin and hemagglutinin with human erythrocytes in acid and alkaline solutiona Strain
pH
Expt 1 C-Stockholm 6.0 6.0 6.0 6.0 8.0 8.0 8.0 8.0 D-1873 6.0 6.0
No. of adsorption Adsorption Start 1 1 3 3 1 1 3 3 Start 3 3
+ + + +
+
HA titer
256 128
