High-Molecular-Weight Hemolysin of Clostridium tetani

INFECTION AND IMMUNITY, Mar. 1982, p. 1086-1090 0019-9567/82/031086-05$02.00/0 Vol. 35, No. 3 High-Molecular-Weight Hemolysin of Clostridium tetani ...
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INFECTION AND IMMUNITY, Mar. 1982, p. 1086-1090 0019-9567/82/031086-05$02.00/0

Vol. 35, No. 3

High-Molecular-Weight Hemolysin of Clostridium tetani KEN'ICHIRO MITSUI,* NORIKO MITSUI, KYOICHI KOBASHI, AND JUN'ICHI HASE Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama-shi 930-01, Japan

Received 31 July 1981/Accepted 21 October 1981

Clostridium tetani excretes hemolysins of two size classes, a high-molecularweight hemolysin (HMH), which was eluted near void volume of a Sepharose 6B column, and conventional tetanolysin (molecular weight, approximately 50,000). The total hemolysin activity in the culture supernatant increased sharply with growth of bacteria and remained at a high level during autolysis. The content of HMH, however, decreased from 41% at 4 h of culture to 0.4% at the early stage of autolysis. The cell bodies also exhibited hemolytic activity, 70% of which could be solubilized and separated into HMH and the 50,000 Mr tetanolysin as extracellular hemolysins. The activity ratio of HMH to the total solubilized hemolysins was 0.45, on the average, at 6 h of culture but was 0.23 at the middle of logarithmic growth. Partially purified HMH from both sources appeared as broken pieces of cytoplasmic membranes under an electron microscope. The ratio of proteins to phospholipids in HMH was found to be 3.26, a value similar to that in cell membrane. The total cell hemolytic activity decreased by 90 or 75% upon addition of chloramphenicol or anti-tetanolysin serum, respectively, into a 6-h-old culture of bacteria. It is suggested that HMH is a complex of tetanolysin with a membrane fragment and releases the conventional tetanolysin during bacterial culture. In an earlier report (9), we demonstrated the presence of two molecular weight classes of hemolysins excreted by Clostridium tetani. Tetanolysin, the smaller of the two, was composed of four electrophoretically different species with molecular weights ranging from 48,000 to 53,000. The other hemolysin was found in the void volume of a Sephadex G-100 column as the minor peak, with an apparent molecular weight of greater than 100,000. The presence of highmolecular-weight hemolysin (HMH) similar to this in the culture fluid had not been reported previously. To understand the mode of formation of HMH and a possible precursor-product relationship between the two classes of hemolysins, we studied the properties and distributions of HMH at various stages of bacterial growth. The results suggest that HMH is a complex of hemolysin with a membrane lipid from which the conventional tetanolysin is released during autolysis.

(grade VI) were products of Sigma Chemical Co. Chloramphenicol was obtained from Sankyo Pharmaceutical Co., Japan. All of the other chemicals were purchased from Nakarai Chemical Co., Japan, and were of analytical reagent grade. Culture of bacteria. The procedure of cultivation was as described previously (9), except proteose peptone replaced 3% (wt/vol) Trypticase in culture medium. The cells were harvested at the indicated time by centrifugation at 8,000 x g for 20 min. After two rinses with cold saline, the cells were suspended in saline. Sonication of cells. The cells were disrupted by ultrasonic vibration with a Kontes Sonifier at 8 A (Kontes Inc., Vineland, N.J.) for three 2-min intervals. Sonication was interrupted to maintain the temperature below 10°C. After sonication, the mixtures were centrifuged at 25,000 x g for 15 min, and hemolytic activity in the supernatants or cell debris was measured. Lysozyme treatment of cells. To the cell suspensions in phosphate-buffered saline (70 mM phosphate buffer [pH 6.8]-78 mM NaCI) containing 0.3 M sucrose was added egg white lysozyme to a final concentration of 0.05%, and the mixture was incubated at 37°C for 30

MATERIALS AND METHODS Materials. C. tetani, strain Harvard A-47, stocked in liver-liver broth, was kindly provided by S. Nishida, School of Medicine, Kanazawa University, City, Japan. Anti-tetanolysin serum was kindly supplied by A. W. Bemheimer, New York University, School of Medicine, New York. Trypticase and yeast extracts were products of BBL Microbiology Systems and Difco Laboratories, respectively. Bovine serum albumin (fraction V) and chicken egg white lysozyme

min.

Gel filtration. A sample was applied onto a Sephadex G-100 or Sepharose 6B column (3.0 by 60 cm), equilibrated with phosphate-buffered saline, and then eluted with the same buffer. Fractions of 5 ml were collected at a flow rate of 10 mlIh. Assay methods. Before assay of activities, samples were pretreated with L-cysteine (2, 8). The hemolytic activity was measured by the method of Roth and Pillemer (11), with a slight modification, using sheep erythrocytes (8). The activity which lyses half of the

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TABLE 1. Activity ratio of extracellular HMH to total extracellular hemolysins Culture time Culture filtrate

(h)

0

20

40

80

60

CULTURE TIME

100

h

FIG. 1. Production of extracellular hemolysins and neurotoxin (lethal activity) during the bacterial culture.

cells in 3 ml of a 1% (vol/vol) cell suspension was defined as one hemolytic unit (HU). Neurotoxin was assayed by the mouse lethality assay of Hardegree (1). Protein concentrations were measured by the method of Lowry et al. (6), using bovine serum albumin (fraction V) as the standard. Organic phosphate in HMH was determined by the method of Martin and Doty (7).

RESULTS

Extracellular hemolysins. The extracellular hemolytic activity rapidly increased with growth until autolysis began. During autolysis, the hemolytic activity reached a plateau and remained at the same level for up to 60 h (Fig. 1). However, the lethal activity (neurotoxin) appeared only at the onset of autolysis and increased over several days. The extracellular hemolysins could be separated into fractions by gel filtration. The first peak corresponded to the extracellular HMH, and the second corresponded to the conventional tetanolysin (Fig. 2),

(HU/ml)

HMH/total

activity rto (%)a 41

HMH activity

(HU/MI)b

4 21 8.4 6 92 23 20.6 8 195 8 16.1 10 438 3.8 16.7 12 585 2.7 16.0 14 585 2.5 14.4 24 668 0.4 2.7 a A sample of the culture filtrate was applied onto a Sephadex G-100 column to separate HMH and tetanolysin. The activity ratio was calculated from the total HU of the two fractions. b Concentration of HMH contained in the culture filtrate.

reported earlier (9). The concentration of extracellular HMH increased to a maximum level by 6 h and then decreased 90% by 24 h (Table 1). Release of hemolysins from C. tetani cells. Washed cells in suspension exhibited hemolytic activity (Table 2). About 90% of the activity was solubilized by washing with saline, followed by sonication. The cell-associated hemolytic activity was the highest at 10 h of culture and then decreased with time to zero after 60 h (Fig. 3). The solubilized hemolysins were also found to separate into two fractions differing in apparent molecular weights on gel filtration. The activity ratio of the cell-bound HMH to the total hemolysins was 45 and 25%, on the average, at 6 and 8 to 11 h of culture, respectively (Table 3). Effect of chloramphenicol or anti-tetanolysin serum. Upon the addition of chloramphenicol after 6 h of culture, the cell-bound hemolytic activity decreased to less than 8% in 30 min, whereas the excreted hemolytic activity was not affected at all (Table 4). When the anti-tetanolyas

).4 ° Es

F.L

El

0

Eq

uz

m

2: X

E-

FRACTION NUMBER

FIG. 2. Sephadex G-100 gel filtration profile of the excreted hemolysin obtained at 6 h of culture.

TABLE 2. Release of hemolysin from cell bodiesa Supernatants Cells Saline wash Sonication (HU) (HU)b (HU)c (HU) 6 200 151 42 16,800 12 359 244 90 30,500 24 35 31 0 37,000 48 20 17 0 30,000 a The cells and supernatants were separated by centrifugation at the indicated culture times. The cells were washed seven times with saline, suspended in saline, and then sonicated. b The hemolytic activity of the cells was measured with a sample of the cell suspension. c Combined saline extracts. Culture time (h)

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TABLE 3. Percentage of HMH in the total solubilized hemolysin from cells by sonication

-20

a 30

t)

E-

Culture time (h)

6b 8b

_H

20

El

.10 ffi.4

ah X

OEh

10

Ed

0

20

40

60

% Recovery'

(%)a

H

u

HMH/total activity ratio

45 27 23

87 72 77

11 After sonication, the supernatants obtained by centrifugation were separated on a Sephadex G-100 column for the determination of the activity ratio. b Average of three experiments. c Total recovery of activity from the column. a

CULTURE TIME ( h )

FIG. 3. Relationship between the culture time and the cell-bound hemolytic activity solubilized by sonication.

sin serum was added, the cell-bound hemolytic activity decreased to 26% in 30 min and could not be extracted with saline. All hemolytic activity in the culture fluid was neutralized by antiserum (Table 4), suggesting that both HMH and the conventional tetanolysin reacted with antitetanolysin. Characterization of extracellular and cellbound HMH. Both HMHs were eluted near the void volume of the Sepharose 6B column, suggesting that their molecular weights were greater than that of thyroglobulin (molecular weight, 669,000 [Fig. 4]). These fractions were further purified by ultracentrifugation (40,000 x g, 60 min), followed by sucrose gradient centrifugation of the supernatants. Rechromatograms on Sepharose 6B of most active fractions obtained by sucrose gradient ultracentrifugation showed a single peak in the same position (Fig. 4). The specific hemolytic activities of the extracellular and cell-bound HMH were 10,000 and 9,400 HU/ mg of protein, respectively. Both HMH fractions contained proteins and phospholipids at a weight ratio of 3.26:1.00, assuming that membrane phospholipids are composed of only dipalmitoyl glycerophosphocholine. Electron micro-

scopic observation of the purified HMH from the cell-bound and extracellular fractions revealed a homogeneous particlate structure which seemed to be small fragments of the cytoplasmic membranes (Fig. 5). DISCUSSION Electron microscopic observation revealed that HMH from both the culture supernatant and cell bodies appeared to be composed of cytoplasmic membrane particles. Indeed, the protein-to-phospholipid weight ratio in the HMH preparation was found to be 3.26:1.00, which was very close to the ratio (3.09:1.00) reported by Korn (4) that was calculated from amounts of amino acids and phospholipids in cell membranes of some gram-positive bacteria. This fact suggests that HMH is a cell membrane-bound hemolysin. This suggestion is further supported by the observation that, on addition of chloramphenicol or antiserum which stops protein synthesis or neutralizes tetanolysin, hemolysin could not be extracted from the cells by washing (Table 4). Based on the cellular location of bacteria toxins described by Raynaud and Alouf (10), conventional tetanolysin, like other oxygen-labile hemolysins, is classified in group III (true exotoxins). Most of the hemolysin produced by C. tetani was, indeed, excreted into the sur-

TABLE 4. Effect of addition of chloramphenicol or anti-tetanolysin serum on the hemolytic activity in the cells Saline wash (HU)Y Cells (HU) Culture filtrate (HU) Expt Addition Disrupted cells (HU)b 730 265 None 455 42,300 24 1 0 Chloramphenicol 40,000 23 190 0 0 Antiserum 197 706 None 40,000 1,220 1,086 52 100 56 Chloramphenicol 40,000 a Cell bodies were washed four times with saline, and the total hemolytic activity in the washing

2

determined. b Washed cells were sonicated in experiment 1 and were treated with lysozyme in

experiment 2.

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FIG. 4. Sepharose 6B gel filtration profile of the cell-bound hemolysin (a) and the extracellular HMH fraction (b). After cultivation for 6 h, the cultures were centrifuged at 8,000 x g for 20 min. (a) The cells were washed with saline and disrupted by repeated freezing and thawing and then sonication. After centrifugation, ammonium sulfate was added to the supernatants to 60% saturation. The precipitate obtained was dissolved in saline and applied onto a Sepharose 6B column. (b) The culture supernatnat was made 60% saturation with ammonium sulfate. Ammonium sulfate precipitation was repeated once, and the precipitate was dissolved in saline for gel filtration on a Sephadex G-100 column to obtain the HMH fraction. This was concentrated by ultrafiltration to apply onto a Sepharose 6B column.

FIG. 5. Electron microscopic image of the cellbound HMH after negative stain. Electron microscopy of the extracellular HMH was identical.

ly transudes through limited sites on the cell wall, some of which are attached and excreted with fragments of cell membrane as HMH, and is converted to conventional tetanolysin later in the culture. An additional indication to this hypothesis was that both HMH and conventional tetanolysin were completely neutralized by anti-tetanolysin serum, suggesting an identical immunoreactivity for both hemolysins. However, attempts to cause conversion of HMH to conventional tetanolysin by proteolysis with trypsin or a crude protease preparation of C. tetani (3) were unsuccessful.

rounding medium during the logarithmic phase (Fig. 1). In contrast, pneumolysin, which has long been known to be cell-associated hemolysin (12, 13), is very often purified by cell washing. However, cell membrane-bound hemolysin of Streptococcus pneumoniae has been reported to have a molecular weight of 63,000 (5). To our knowledge, hemolysins of high molecular weights, such as the HMH of C. tetani, have not been reported from either a cell-bound or an extracellular source. ACKNOWLEDGMIENTS The cell-bound hemolytic activity reached a We are indebted to Chun-Yen Lai and K. lwata, Roche maximum at 10 h of culture and then diminished Institute of Molecular Biology, for their valuable suggestions to zero after 60 h. In contrast, the extracellular and T. Yasuda, Institute of Medical Sciences, University of hemolytic activity increased with growth to 20 h Tokyo, for the electron microscopy. of culture (stationary phase) and then slightly increased to a plateau during autolysis. This LITERATURE CITED slight increase and maintenance of activity for 80 1. M. C. 1965. Separation of neurotoxin and Hardegree, h are considered to be owing possibly to the hemolysin of Clostridium tetani. Proc. Soc. Exp. Biol. release of cell membrane-bound hemolysins. Med. 119:405-408. The percentage of HMH in both culture super- 2. Hase, J., K. Mfitsui, and E. Shonaka. 1975. Clostridium perfringens exotoxins III. Binding of 0-toxin to erythronatant and cell-bound hemolysins decreased cyte membranes. Jpn J. Exp. Med. 45:433-438. rapidly even before autolysis began (Tables 1 3. Helting, T. B., S. Parschat, and H. Engelhardt. 1979. and 3). These results suggest the possibility that, Structure of tetanus toxin: demonstration and separation of a specific enzyme converting intracellular tetanus toxin at an early stage of growth, tetanolysin vigorous-

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to the extracellular form. J. Biol. Chem. 254:10728-10733. 4. Korn, E. D. 1966. Structure of biological membranes. Science 153:1491-1498. 5. Kreger, A. S., and A. W. Bernheimer. 1969. Physical behavior of pneumolysin. J. Bacteriol. 98:306-307. 6. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Martin, J. B., and D. M. Doty. 1949. Determination of inorganic phosphate: modification of isobutyl alcohol procedure. Anal. Chem. 21:965-967. 8. Mitsui, K., N. Mitsui, and J. Hase. 1973. Clostridium perfringens exotoxins II. Purification and some properties of 0-toxin. Jpn J. Exp. Med. 43:377-391. 9. Mitsui, N., K. Mitsui, and J. Hase. 1980. Purification and

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11.

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some properties of tetanolysin. Microbiol. Immunol. 24:575-584. Raynaud, M., and J. E. Alouf. 1960. Intracellular versus extracellular toxins, p. 67-112. In S. J. Ajl, S. Kadis, and T. C. Montie (ed.), Microbial toxins. Academic Press, Inc., New York. Roth, F. B., and L. Pillemer. 1955. Purification and some properties of Clostridium welchii type A theta toxin. J. Immunol. 75:50-56. Smyth, C. J., and J. L. Duncan. 1978. Thiol-activated (oxygen-labile) cytolysins, p. 129-183. In J. Jeljaszewicz and T. Wedstrom (ed.), Bacterial toxins and cell membranes. Academic Press, Inc., New York. Shumway, C. N., and S. J. Klebanoff. 1971. Purification of pneumolysin. Infect. Immun. 4:388-392.