International Journal of Food Microbiology, 10 ( 1 9 9 0 ) 1 1 3 - 1 2 4 Elsevier
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F O O D 80007
Clostridium botulinum toxins A.H.W.
Hauschild
Microbiology Research Division, Health Protection Branch, Ottawa, Ontario, Canada
Structure, activation, destruction, lethality, genesis, neurotoxic action and medical application of botulinal toxins are reviewed. Key words: Clostridium botulinum; Toxins; Foods
Introduction Eight distinct toxins of Clostridium botulinum designated A, B, C 1, C z, D, E, F and G, are presently recognized. They are all proteins made up of about 20 c o m m o n amino acids and, with the exception of toxin C z, exhibit a characteristic neurotoxic action. Toxins are formed by each of the four diverse groups of C. botulinum. In addition, recent isolates from cases of infant botulism, identified as C. butyricum and C. barati, were found to produce botulinal toxins E (Aureli et al., 1986) and F (Hall et al., 1985), respectively (Table I). In general, individual strains of C. botulinum produce a single toxin. Exceptions are some rare isolates of group I, each producing one major and one minor toxin (Gim6nez and Ciccarelli, 1978; Hatheway et al., 1981; McCroskey and Hatheway,
TABLE I
Clostridia producing botulinal toxins Producer species
Group
Toxin type
C. botulinum
1 II III IV
A, B, F B,E,F C I , C 2, D G
C. butyricum
E
C. barati
F
Correspondence address: Microbiology Research Division; Health Protection Branch, Ottawa, Ontario, Canada K 1 A 0 L 2 . 0 1 6 8 - 1 6 0 5 / 9 0 / $ 0 3 . 5 0 ~ 1990 Elsevier Science Publishers B.V.
114 T A B L E 11 T o x i n s of CIostridium botulinum
C. botulinum
T o x i n type
Group
Type
1
A
Subtype
Major
Minor
AB AF
A A A
B F
B
F
B B B F
II
B E F
B E F
-
I11
C
(71
C 2, D C2 C t, C~
BA BF
IV
C~ C/~
D
D
G
G
A F
1984; Poumeyrol et al., 1983), and strains of types C and D which may form up to three toxins each (Eklund and Poysky, 1981) (Table II). The type designation of strains is identical to that of their major toxin. Toxins and antitoxins of types B and F each cross-neutralize completely between groups I and II, but the type-specific toxins of the two groups are not identic'a_l (Shimizu et al., 1974). Rymkiewicz et al. (1979) reported antitoxin titers for sera to group II toxin B that were six times higher in titrations of homologous toxin than in titrations of group I toxin B. Toxins C and D and their antitoxins also cross-react (Oguma et al., 1980), and antitoxin E is capable of neutralizing small quantities of toxin F (Eklund et al., 1967). Botulinal toxins are produced during growth, but little toxin is released from the intact cell. The main release of toxin starts at the end of the growth phase and coincides with cell lysis (Duda and Slack, 1969; Siegel and Metzger, 1979).
Structure The botulinal toxins may exist in four molecular sizes of approximately 7S, 12S, 16S and 19S, designated S (small), M (medium), L (large) and LL (extra large) toxins, respectively (Sakaguchi et al., 1981). The molecular weights range from 150 to 900 kDa (DasGupta, 1981; Sugiyama, 1980). Only the M(12S) and L(16S) forms are encountered in toxic foods and cultures (Hauschild and Hilsheimer, 1969; Sugii and Sakaguchi, 1977). Of these, the M form is the more c o m m o n natural toxin. It is an aggregate of a toxic component, the small (7S) single-chain toxin, with an atoxic
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component. The L toxin also includes a haemagglutinin in the complex. Natural toxins (M and L) are commonly referred to as 'progenitor toxins', and S toxin is referred to as 'derivative toxin' (Lamanna and Sakaguchi, 1971). LL toxin was the first botulinal toxin to be purified and crystallized (Lamanna and Carr, 1967) and is only known for type A. This toxin has not been encountered in cultures and is likely an artificial aggregate. Under mild alkaline conditions, progenitor toxins dissociate into S toxin and atoxic components which can be separated by ion exchange chromatography (Boroff and DasGupta, 1971). The derivative (S) toxin, a single-chain protein, is 'nicked' by a number of proteases, including proteases of group I C. botulinum, into a dichain molecule, with the two chains remaining associated by a disulfide bond. This bond may be severed by reducing agents, e.g., dithiothreitol. Only toxin C 2 has no covalent linkage between the two chains. The two components of nicked toxin, a light (L) and heavy (H) chain, have molecular weights of roughly 50 and 100 kDa, respectively, and can be separated by polyacrylamide gel electrophoresis (Sugiyama, 1980). Individually, the two components are nontoxic, but toxicity can be restored by reestablishing the covalent bond through oxidation. Toxin C 2 activity can be restored by simply mixing the two chains (Ohishi et al., 1980). The L and H chains are antigenically distinct, and antibodies to either of the two, particularly the H component, may neutralize the complete toxin (Kozaki et al., 1986; Syoto and Kubo, 1981). Toxins A, B~ C 1, C 2, D, E and F have been purified and characterized. They all have basically the same structure, with molecular weights of the S toxin, L and H chains in the order of 150, 50 and 100 kDa, respectively. Estimates based on amino acid composition were essentially the same ( D a s G u p t a and Woody, 1984).
Activation
The lethality of progenitor and derivative toxins can be increased by a few log10 units in the presence of proteolytic enzymes. Group II toxins (B, E and F) and toxins C 2 and G are particularly susceptible to activation (Eklund and Poysky, 1972; Iida, 1968; Solomon et al., 1985). Yokosawa et al. (1986) reported 30-fold higher activation of toxin E extracted from cells as compared to the toxin in culture fluid, which indicates that some activation takes place also in 'non-proteolytic' group II cultures. Trypsin activation of botulinal toxin is accompanied by conversion of single-chain toxin to nicked toxin. However, the process of nicking seems irrelevant to the mechanism of activation because activation with trypsin at a low pH (4.5) or with a 'trypsin-like enzyme' (TLE) from a proteolytic type B culture has been shown to proceed without a significant degree of nicking ( D a s G u p t a and Sugiyama, 1976; Ohishi and Sakaguchi, 1977). The relevant cleavage associated with activation occurs on the H chain (Ohishi et al., 1980) and likely involves the hydrolysis of a lysyl bond (Miura, 1974). The cleavage site remains obscure. D a s G u p t a and Sugiyama (1976) found no detectable
116
change in molecular structure as a result of toxin E activation with TLE, while Ohishi (1987) reported a molecular weight change from 101 kDa to 88 kDa in the trypsin-activated H (II) component of toxin C 2.
Inactivation
Two aspects of toxin inactivation are of particular interest: the thermal destruction of toxins in foods, and their destruction in drinking water at ambient temperatures. Thermal inactivation of botulinal toxins is nonlinear. The destruction curves are generally diphasic, with an initial steep decline levelling off with time (Cartwright and Lauffer, 1958; Woodburn et al., 1979). The curves may be explained by differences in the thermostability of progenitor toxin, variation in the degree of hydrogen bonding, or aggregation during heating (Lamanna, 1959; Licciardello et al., 1967b). The nonlinear heat inactivation curves preclude the expression of thermoresistance in D-values. Instead, the heat resistance of botulinal toxins is expressed as the time required for a given toxin concentration (Co) to be reduced to a single LDs0 dose ( F value). With Co of about 1 0 4 m o u s e LDs0/g, a level often encountered in toxic foods, F values for toxins A, B, E, F and G in a variety of foods at 78-80 ° C varied from < 1 min to 2 min (Bradshaw et al., 1979, 1981; Licciardello, 1967a). With Co of about 105 LDs0/g, F values at 79 o C ranged from 2 to 20 min for toxin A, from 1.5 to 10 min for toxin B and from 1.5 to 5 rain for toxin E (Woodburn et al., 1979). Relatively high F values have been recorded in acid foods such as tomato juice. This is consistent with maximum thermoresistance of botulinal toxins at pH 4-5. F values are increased also in the presence of proteins and colloidal materials and by increased levels of ionic strength. Consequently, foods are generally more protective than buffers (Bradshaw et al., 1979, 1981; Woodburn et al., 1979; Woolford et al., 1978). For safe thermal inactivation of botulinal toxins at levels up to 10 s LDs0/g in foods, Woodburn et al. (1979) recommended time/temperature combinations of 20 min at 7 0 ° C or 5 min at 85 °C. In contrast to the nonlinear inactivation of toxin with time, F values plotted against temperature follow a straight line. Their slopes indicate Z-values of 3-5 ° C for toxins A and B, 4 - 6 ° C for toxins F and G, and 7 8 ° C for toxin E (Bradshaw et ai., 1979, 1981; Licciardello, 1967a). In spite of its significance, the question of toxin stability in drinking water has rarely been addressed experimentally. Toxin inactivation in water is nonlinear and, like heat destruction, slows down with time. Schantz (personal communication) observed a 2.5 log10 decrease of toxin A in drinking water after 2 days, but a total decrease of only 3.5 log m units after 6 days. Relative toxin stability in water has also been reported by others (Brygoo, 1953; Markarjan et al., 1960). For rapid detoxification of drinking water within 1 h, about 10 mg of residual chlorine per 1
117
would be required. Alternative means of toxin removal involve alum treatment and filtration, or active charcoal treatment.
Lethality When botulinal toxins are administered by the parenteral route, the atoxic component has little or no effect on toxicity (Sakaguchi et al., 1984). In contrast, oral toxicities increase by up to 1 0 4 log10 units with increasing molecular weight of the toxin indicating that the atoxic moiety protects the toxic component in the digestive tract (Ohishi et al., 1977; Sakaguchi et al., 1981). Protection of progenitor toxin from gastric juice or pepsin by its atoxic component has been demonstrated in vitro (Sakaguchi et al., 1981). The protective role of the atoxic component ends with the toxin uptake into the lymph which occurs predominantly in the duodenum and jejunum (Ohishi, 1983a). Before entering the lymph, the atoxic portion is shed, and only the 7 S form is encountered in the lymph and blood stream (Bonventre, 1979; Hildebrand et al., 1961). Lethal doses of ingested toxin for humans are estimated as the toxin content of the food consumed by fatal or near-fatal cases of botulism without effective treatment. Because of uncertainties about the amount of toxin ingested and variations in the proportions of L and M toxins among toxic foods, such estimates can only be approximate. These range from about 5 × 10 3 tO 1 × 105 mouse (i.p.) LDs0 for group I toxins A and B (Bonventre, 1979; Meyer and Eddie, 1951; Morton, 1961) and from 1 ×105 to 5 × 1 0 s mouse LDs0 for group II toxins B and E (Dolman et al., 1955; Ralovich and Barna, 1966). The lethality of toxin F in humans is likely much lower. In monkeys, the oral lethal dose of group I toxin F was 5 × 104 mouse LDs0/kg (Dolman and Murakami, 1961) which is comparable to the lethality of toxin G (Ciccaretli et al., 1977) but more than a log m unit lower than that of toxins A, B and E (0.5-2.5 x 103 mouse LDs0/kg ) (Smith, 1977; Dolman and Murakami, 1961).
Genetics of toxigenesis A number of bacterial protein toxins are specified by plasmids or bacteriophages (Murphy et al., 1984; H~irtlein et al., 1984). Plasmids and bacteriophages have been demonstrated in nearly all types of C. botulinum, but no association between botulinal toxigenesis and plasmids has been found (Strom et al., 1984; Weickert et al., 1986), and mediation of toxin production by bacteriophages has been established only for toxins C 1 and D of C. botulinum types C and D. The bacteriophages of these types are characterized by hexagonal heads and long, partially sheathed tails of up to 450 nm (Eklund et al., 1972; Inoue and Iida, 1968). Bacteriophages of other C. botulinum types show similar structures.
118 Loss of bacteriophages by toxigenic strains of types C and D, e.g., by repeated transfer, ultraviolet irradiation or acridine orange treatment, is accompanied by the loss of ability to produce C~ and D toxins, and loss of resistance to lysis by bacteriophages of the toxic parent strain or related phages. This process is reversed when nontoxigenic cultures are reinfected with specific bacteriophages. Both toxins of a given strain are controlled by a single phage (Eklund and Poysky, 1981; Inoue and Iida, 1970). Some strains of types C and D may be converted from one type to another by curing them of their prophages and reinfecting them with viruses from types D and C, respectively (Eklund and Poysky, 1974, 1981). Eklund et al. (1974) also demonstrated compatibility between strains and bacteriophages of different species. A strain of type C, cured of its prophage and reinfected with phage NA1 of Clostridiurn novyi type A, also gained the ability to produce C. novyi alpha toxin. These conversions are all reversible. Control of toxigenesis by bacteriophages has not been established for other types of C. botulinum. In case of a stable host-phage relationship, such a control might be difficult to demonstrate. In strains of types C and D, this relationship is generally unstable as indicated by the loss of phages after transfers. This loss is even accelerated when antiserum against the host-specific phage is added to growing cultures to avoid reinfection of phage-cured cells (Eklund and Poysky, 1981: Iida et al., 1981).
Neurotoxic action
With the exception of toxin C 2, the botulinal toxins exhibit a specific neurotoxic action which results in the blockage of transmitter release at the neuromuscular junctions. Both cholinergic and adrenergic systems are affected, but much higher concentrations are required to inhibit the release of noradrenaline than that of acetyl choline (Habermann, 1981; Simpson, 1979). Three stages in the neurotoxic mechanism have been recognized: toxin binding, toxin internalization and blockage of transmitter release (Black and Dolly, 1986b; Simpson, 1981a). The first step involves the interaction of toxin with specific receptor sites on the presynaptic membrane which is mediated by the H chain (Bandyopadhyay et al., 1987; Black and Dolly, 1986a, 1986b; Kitamura, 1976). The receptor likely contains sialic acid as an essential ingredient because neuraminidase and gangliosides both interfere with toxin binding (Bigalke et al., 1986; Williams et al., 1983), and gangliosides are potent toxin receptors (Ochanda et al., 1986; Takamizawa et al., 1986). The binding sites are not the same for each toxin, e.g., binding of toxin A to synaptosomes is in competition with toxin E but not with toxin B (Kozaki, 1979). The second step involves translocation of the toxin, or a fraction of it, through the presynaptic membrane (Black and Dolly, 1986a; Donovan and Middlebrook, 1986). The process only occurs after binding to the receptor and is energy (ATP) and temperature dependent (Black and Dolly, 1986b; Simpson, 1981b). From here on, the toxin can no longer be neutralized (Simpson, 1981a).
119 The final stage involves the blockage of neurotransmitter release. Since diffeeent transmitters are affected, the action site is likely the same for all transmitter systems (Bigalke et al., 1985). Pumplin and Reese (1977) demonstrated interference of botulinal toxin in the fusion of acetyl choline containing cytoplasmic vesicles with the presynaptic membrane. It has long been postulated that the underlying toxin reaction is enzymatic (Simpson, 1981b). Ohashi and Narumiya (1987a) and Ohashi et al. (1987b) reported enzymatic A D P ribosyltransferase activity of C1 and D toxins involving a membrane protein with a molecular weight of 21 kDa: N A D + protein (21 kDa) ~ ADP-ribose-protein + nicotinamide. The authors suggested that the neurotoxins may catalyze the transfer of ADPribose from N A D o a specific membrane protein involved in the exocytosis of transmitter (Ohashi and Narumiya, 1987a). Matsuoka et al. (1987) confirmed the enzymatic activity of toxins C1 and D, except that the protein substrates had slightly larger molecular weights (24-26 kDa). Aktories et al. (1987) reported ribosyl transferase activity of C. botulinum type C with a 21-kDa protein as substrate, but the activity was associated with a low-molecular weight (24 kDa) enzyme which could not be toxin C 1. No enzymic activity has been demonstrated for any of the other neurotoxins.
Action of toxin C 2
Toxin C 2 differs from other botulinal toxins in the absence of a covalent bond between L and H chains, its lack of neurotoxicity, and a unique pathological mechanism manifesting itself in the leakage of plasma protein into the intestinal lumen, intestinal fluid accumulation, vacuolization of epithelial cells, intercellular edema, necrosis of the mucosa, increased vascular permeability, hypotension, and hemorrhaging of the lungs (Aktories et al., 1986a; Ohishi and Ogadiri, 1984; Simpson, 1982, 1984). Toxin C 2 ribosylates a specific protein of 43 kDa which was identified as G-actin (Aktories et al., 1986a, 1986b; Ohishi, 1986). As a consequence of actin binding, the microfilament network structure of the host cell becomes disorganized (Reuner et al., 1987). The ribosyltransferase activity resides in the L chain, whereas the specific binding is associated with the H chain (Aktories, 1986a; Ohishi, 1983b, 1986).
Beneficial effects of botulinal toxins
This review would be incomplete without the mention of some medical applications of botulinal toxin. Small doses of toxin A in the order of 100 mouse M L D have been used for several years in the treatment of strabismus as an alternative to surgery. Injection of excessively pulling muscles with toxip ~auses temporary local
120
paralysis and results in long-lasting muscle realignments (Magoon and Scott, 1987: Schantz and Scott, 1981). Similarly, eyelid spasms (Cohen et al., 1986) and closures (Katz and Rosenberg, 1987), spasmodic dysphonia (Miller et al., 1987) and urethal sphincter spasticity (Dykstra et al., 1986) have been treated successfully, though with transient relief.
References Aktories, K., B~irmann, M., Ohishi, I., Tsuyama, S., Jakobs, K.H. and Habermann, E. (1986a) Botulinum C2 toxin ADP-ribosylates actin. Nature 322, 390-392. Aktories, K., Ankenbauer, T., Schering, B. and Jakobs, K.H. (1986b) ADP-ribosylation of platelet actin by botulinum C2 toxin. Eur. J. Biochem. 161, 155-162. Aktories, K., Weller, U. and Chhatwal, G.S., (1987) Clostridiurn botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 212, 109-113. Aureli, P., Fenecia, L., Pasolini, B., Bianfranceschi, M., McCroskey, L.M. and Hatheway, C.L. (1986) Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J. Infect. Dis. 154, 207-211. Bandyopadhyay, S., Clark, A.W., DasGupta, B.R. and Sathyamoorthy, V. (1987) Role of the heavy and light chains of botulinum neurotoxin in neuromuscular paralysis. J. Biol. Chem. 262, 2660 2663. Bigalke, H., Dreyer, F. and Bergey, G. (1985) Botulinum A neurotoxin inhibits non-cholinergic synaptic transmission in mouse spinal cord neurons in culture. Brain Res. 360, 318-324. Bigalke, H., Mi~ller, H. and Dreyer, F. (1986) Botulinum A neurotoxin unlike tetanus toxin acts via a neuraminidase sensitive structure. Toxicon 24, 1065-1074. Black, J.D. and Dolly, J.O. (1986a) Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. UItrastructural autoradiographic localization and quantitation of distinct membrane acceptors of types A and B on motor nerves. J. Cell Biol, 103, 521 534. Black, J.D. and Dolly, J.O. (1986b) Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis. J. Cell Biol. 103, 535-544. Bonventre, P.F. (1979) Absorption of botulinal toxin from the gastrointestinal tract. Rev. Infect. Dis. 1, 663 667. Boroff, D.A. and DasGupta, B.R. (1971) Botulinum toxin. In: S. Kadis, T.C. Montie and S.J. Ajl (Eds.), Microbial Toxins. It A. Bacterial protein toxins. Academic Press, New York, pp. 1-68. Bradshaw, J.G.. Peeler, J.T. and Twedt, R.M. (1979) Thermal inactivation of CIostridiurn botulinum toxins A and B in buffer, and beef and mushroom patties. J. Food Sci. 44, 1653-1667. Bradshaw, J.G., Peeler, J.T. and Twedt, R.M. (1981) Thermal inactivation of Clostridiurn botulinum toxin types F and G in buffer and in beef and mushroom patties. J. Food Sci. 46, 688-690, 696. Brygoo, E.-R. (1953) R~sistance des toxines botuliques en dilution dans l'eau. Ann. Inst. Pasteur 84, 1039-1040. Cartwright, T.E. and Lauffer, M.A. (1958) Temperature effect on botulinum A toxin. Proc. Soc. Exp. Biol. Med. 98, 327-330. Ciccarelli, A.S., Whaley, D.N., McCroskey, L.M., Gim6nez, D.F., Dowell, V.R. and Hatheway, C.L. (1977) Cultural and physiological characteristics of CIostridium botulinum type G and the susceptibility of certain animals to its toxin. Appl. Environ. Microbiol. 34, 843-848. Cohen, D.A., Savino, P.J., Stern, M.B. and Hurtig, H.I. (1986) Botulinum injection therapy for blepharospasm: a review and report of 75 patients. Clin. Neuropharmacol. 9, 415-429. DasGupta, B.R. (1981) Structure and structure function relation of botulinum neurotoxins. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press, New York, pp. 1-19. DasGupta, B.R. and Sugiyama, H. (1976) Molecular forms of neurotoxins in proteolytic CIostridium botulinum type B cultures. Infect. lmmun. 14, 680-686.
121 DasGupta, B.R. and Woody, M.A. (1984) Amino acid composition of Clostridium botulinum type A neurotoxin. Toxicon 22, 312-315. Dolman, C.E. and Murakami, L. (1961) Clostridium botulinum type F with recent observations of other types. Infect. Dis. 109, 107-128. Dolman, C.E., Darby, G.E. and Lane, R.F. (1955) Type E botulism due to salmon eggs. Can. J. Publ. Health 46, 135-141. Donovan, J.J. and Middlebrook, J.L. (1986) Ion-conducting channels produced by botulinum toxin in planar lipid membranes. Biochemistry 25, 2872-2876. Duda, J.J. and Slack, J.M. (1969) Toxin production in Clostridium botulinum as demonstrated by electron microscopy. J. Bacteriol. 97, 900-904. Dykstra, D.D., Sisi, A.A., Pagel, J. and Goldish, G. (1986) A study on the effects of botulinum-A toxin to reduce external urethral sphincter spasticity in humans. Arch. Phys. Med. R.ehabil. 67, 681. Eklund, M.W. and Poysky, F.T. (1972) Activation of a toxic component of Clostridium botulinum types C and D by trypsin. Appl. Microbiol. 24, 108-113. Eklund, M.W. and Poysky, F.T. (1974) Interconversion of type C and D strains of Clostridium botulinum by specific bacteriophages. Appl. Microbiol. 27, 251-258. Eklund, M.W. and Poysky, F.T. (1981) Relationship of bacteriophages to the toxigenicity of Clostridium botulinum and closely related organisms. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press, New York, pp. 93-107. Eklund, M.W., Poysky, F.T. and Wider, D.I. (1967) Characteristics of Clostridium botulinum type F isolated from the Pacific coast of the United States. Appl. Microbiol. 15, 1316-1323. Eklund, M.W., Poysky, F.T., Reed, S.M. and Smith, C.A. (1972) Bacteriophage and the toxicity of Clostridium botulinum type C. Science 172, 480-482. Eklund, M.W., Poysky, F.T., Meyers, J.A. and Pelroy, G.A. (1974) Interspecies conversion of CIostridium botulinum type C to Clostridium novyi type A by bacteriophage. Science 186, 456-458. Gimrnez, D.F. and Ciccarelli, A.S. (1978) New strains of Clostridium botulinum subtype Af. Zbl. Bakteriol. Hyg. I Abt. Orig. A 240, 215-220. Habermann, E. (1981) Botulinum A and tetanus toxin--similar actions on transmitter systems in vitro. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press, New York, pp. 129-141. Hall, J.D., McCroskey, L.M., Pincomb, B.J. and Hatheway, C.L. (1985) Isolation of an organism resembling Clostridium barati which produces type F botulinal toxin from an infant with botulism. J. Clin. Microbiol. 21,654-655. Hartlein, M., Hughes, C., Mtiller, D., Kreft, J. and Goebel, W. (1984) Haemolysin genes from gram-negative and gram-positive bacteria. In: J.E. Alouf, F.J. Fehrenbach, J.H. Freer and J. Jeljaszewicz (Eds.), Bacterial Protein Toxins. Academic Press, London, pp. 39-46. Hatheway, C.L., McCroskey, L.M., Lombard, G.L. and Dowell, V.R. (1981) Atypical toxin variant of Clostridium botulinum type B associated with infant botulism. J. Clin. Microbiol. 14, 607-611. Hauschild, A.H.W. and Hilsheimer, R. (1969) Antigenic and chromatographic identity of two apparently distinct toxins of Clostridium botulinum type A. Can. J. Microbiol. 15, 1129-1132. Hildebrand, G.J., Lamanna, C. and Heckley, R.J. (1961) Distribution and particle size of type A botulinum toxin in body fluids of intravenously injected rabbits. Proc. Soc. Exp. Biol. Med. 107, 284-289. lida, H. (1968) Activation of Clostridium botulinum toxin by trypsin. In: M. Herzberg (Ed.), Toxic Microorganisms. Proc. 1st U.S.-Japan Conf. on Toxic Microorganisms, Honolulu, HI, pp. 336-340. lida, H., Oguma, K. and Inoue, K. (1981) Toxin production and phage in Clostridium botulinum types C and D. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press, New York, pp. 109-120. Inoue, K. and lida, H. (1968) Bacteriophages of Clostridium botulinum. J. Virol. 2, 537-540. Inoue, K. and Iida, H. (1970) Conversion of toxigenicity in Clostridium botulinum type C. Jpn. J. Microbiol. 14, 87-89. Katz, B. and Rosenberg, J.H. (1987) Botulinum therapy for apraxia of eyelid opening. Am. J. Ophthalmol. 103, 718-719. Kitamura, J. (1976) Binding of botulinum neurotoxin to the synaptosome fraction of rat brain. Naunyn-Schmiedemeyers. Arch. Pharmacol. 295, 171-175.
122 Kozaki, S, (1979) Interaction of botulinum type A, B and E derivative toxins with synaptosomes of rat brain. Naunyn-Schmiedemeyers. Arch. Pharmacol. 308, 67 70. Kozaki, S., Kamata, Y., Nagai, T., Ogasawara, J. and Sakaguchi. G. (1986) The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin. Infect. lmmun. 52, 786-791. Lamanna, C. (1959) The most poisonous poison. Science 130, 763-772. Lamanna, C. and Carr, C.J. (1967) The botulinal, tetanal and enterostaphylococcal toxins: a review. Clin. Pharmacol. Ther. 8. 286-332. Lamanna, C. and Sakaguchi, G. (1971) Botulinal toxins and the problem of nomenclature of simple toxins. Bacteriol. Rev. 32, 242-249. Licciardello, J.J., Ribich, C.A. Nickerson, J.T.R., and Goldblith, S.A. (1967a) Thermal inactivation of type E botulinum toxin. Appl. Microbiol. 15,249-256. Licciardello, J.J. Ribich, C.A., Nickerson, J.T.R. and Goldblith, S.A. (1967b) Kinetics of the thermal inactivation of type E Clostridium botulinum toxin. Appl. Microbiol. 15, 344 349. Magoon, E. and Scott, A.B. (1987) Botulinum toxin chemodenervation in infants and children: an alternative to incisional strabismus surgery. J. Pediatrics 110, 719 722. Markarjan, M.K., Ryshow, N.V. and Stannikow, J.V. (1960) Sanierung von mit Botulinustoxin verunreinigtem Wasser, J. Hyg. Epidemiol. Microbiol. lmmunol. 4, 385-389. Matsuoka, 1., Syuto, B., Kurihara, K. and Kubo, S. (1987) ADP-ribosylation of specific membrane proteins in pheochromocytoma and primary-cultured brain cells by botulinum neurotoxins type C and D. FEBS Lett. 216. 295-299. McCroskey, L.M. and Hatheway, C.L. (1984) Atypical strains of Clostridiurn botuhnum isolated from specimens of infant botulism cases. Abstr. Annu. Meet., Am. Soc. Microbiol. C159, p. 263. Meyer, K.F. and Eddie, B. (1951) Perspectives concerning botulism. Z. Hyg. lnfektionskr. 133, 255 263. Miller, R.H., Woodson, G.E. and Jankowic, J. (1987) Botulinum toxin injection of the vocal fold for spasmodic dysphonia. Arch. Otolaryngol. Head Neck Surg. 113, 603-605. Miura, T. (1974) Isolation and characterization of an esterase-active enzyme from pronase with special reference to activation of Clostridium botulinum type E progenitor toxin. Jpn. J. Med. Sci. Biol. 27, 285 296. Morton, H.E. (1961) The toxicity of Ch)stridium botulinurn type A toxin for various species of animals, including man. Institute for Cooperative Research, University of Pennsylvania, Philadelphia. Murphy, J.R., Kaczorek, M., Leong, D., Delpeyroux, F., Coleman, K., Chencier, N.. Boquet, P., Streeck, R.E. and Tiollais, P. (1984) The diphtheria toxin structural gene. In: J.E. Alouf, F.J. Fehrenbach, J.H. Freer and J. Jeljaszewicz (Eds.), Bacterial Protein Toxins. Academic Press, London, pp. 29-38. Ochanda, J.O., Syuto, B., Ohishi, I., Naiki, M. and Kubo, S. (1986) Binding of Clostridium hotulinurn neurotoxin to gangliosides. J. Biochem. 100, 27 33. Oguma, K., Syuto, B., lida, H. and Kubo, S. (1980) Antigenic similarity of toxins produced by Clostridium botulinurn type C and D strains. Infect. Immun. 30, 656-660. Ohashi, Y. and Narumiya, S. (1987a) ADP-ribosylation of a M~ 21,000 membrane protein by type D botulinum toxin. J. Biol. Chem. 262, 1430-1433. Ohashi, Y., Kamiya, T., Fujiwara, M. and Narumiya, S. (1987b) ADP-ribosylation by type C l and D botulinum neurotoxins: stimulation by guanine nucleotides and inhibition by guanidino-containing compounds. Biochem. Biophys. Res. Comm. 142, 1032-1038. Ohishi, 1. (1983a) Absorption of Clostridium botulinum type B toxins of different molecular sizes from different regions of rat intestine. FEMS Microbiol. Lett. 16, 257 260. Ohishi, I. (1983b) Response of mouse intestinal loop to botulinum C 2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components. Infect. lmmun. 40, 691-695. Ohishi, 1. (1986) NAD-glycohydrolase activity of botulinum C 2 toxin: a possible role of component I in the mode of action of the toxin. J. Biochem. 100, 407-413. Ohishi, 1. (1987) Activation of botulinum C 2 toxin by trypsin. Infect. Immun. 55, 1461 1465. Ohishi, 1. and Odagiri, Y. (1984) Histopathological effect of botulinum C 2 toxin on mouse intestines. Infect. Immun. 43, 54 58. Ohishi, 1. and Sakaguchi, G. (1977) Activation of botulinum toxins in the absence of nicking. Infect. lmmun. 17, 402-407.
123 Ohishi, 1., Sugii, S. and Sakaguchi, G. (1977) Oral toxicities of Clostridium hotulinum toxins in response to molecular size. Infect. Immun. 16, 107-109. Ohishi, !., Iwasaki, M. and Sakaguchi, G. (1980) Purification and characterization of two components of botulinum C 2 toxin. Infect. Immun. 30, 668-673. Poumeyrol, M., Billon, J., Delille, F., Haas, C., Marmonier, A. and Sebald, M. (1983) Intoxication botylique mortelle due h une souche de CIostridium botulinum de type AB. Mrd. Malad. Infect. 13, 750-754. Pumplin, D.W. and Reese, T.S. (1977) Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze-fracture technique. J. Physiol. 273. 443-457. Ralovich, B. and Barna, K. (1966) Durch toxikologische und bakteriologische Untersuchungen erwiesener Typ B Botulismus. Zbl. Bakteriol. 1 Orig. 200, 509 519. Reuner, K.H., Presek, P., Boschek, C.B. and Aktories, K. (1987) Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells. Europ. J. Cell Biol. 43, 134 140. Rymkiewicz, D., Sawicki, J. and Briihl, A. (1979) Study on the immunological heterogeneity of CTostridium botulinum B type toxin. Arch. lmmunol. Therap. Exp. 27, 709 714. Sakaguchi, G., Ohishi, I. and Kozaki, S. (1981) Purification and oral toxicities of Clostridium hotulinum progenitor toxins. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press, New York, pp. 21 34. Sakaguchi, G., Kozaki, S. and Ohishi, I. (1984) Structure and function of botulinum toxins. In: J.E. Alouf, F.J. Fehrenbach, J.H. Freer and J. Jeljaszewicz (Eds.), Bacterial Protein Toxins. Academic Press, London, pp. 435-443. Schantz, E.J. and Scott, A.B. (1981) Use of crystalline type A botulinum toxin in medical research. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press. New York, pp. 143 150. Shimizu, T., Kondo, H. and Sakaguchi, G. (1974) Immunological heterogeneity of Clostridiurn botulinum type B toxins. Jpn. J. Med. Sci. Biol. 27, 99 100. Siegel, LS. and Metzger, J.F. (1979) Toxin production by CIostridium botulinum type A under various culture conditions. Appl. Environ. Microbiol. 38, 606-611. Simpson, L.L. (1979) The action of botulinal toxin. Rev. Infect. Dis. 1,656 659. Simpson, L.L. (1981a) The origin, structure and pharmacological activity of botulinum toxin. Pharmacol. Rev. 33, 155 188. Simpson, L.L. (1981b) Pharmacological studies of the cellular and subcellular effects of botulinum toxin. In: G.E. Lewis (Ed.), Biomedical Aspects of Botulism. Academic Press. New York, pp. 35-46. Simpson, L.L. (1982) A comparison of the pharmacological properties of C!ostridium botulinum type C I and C 2 toxins. J. Pharmacol. Exp. Therap. 223, 695 701. Simpson, k.k. (1984) Molecular basis for the pharmacological actions of Clostridium botulinum type C 2 toxin. J. Pharmacol. Exp. Therap. 230, 665-669. Smith, L.DS. (1977) Botulism. The organism, its toxins, the disease. C.C. Thomas Publ.. Springfield, Ik. Solomon, H.M., Kautter, D.A. and kynt, R.K. (1985) Common characteristics of the Swiss and Argentine strains of Clostridium botulinum type G. J. Food Protect. 48~ 7-10. Strom, M.S., Eklund, M.W. and Poysky, F.T. (1984) Plasmids in Ch~stridium hotulinum and related Clostridium species. Appl. Environ. Microbiol. 48, 956 963. Sugii, S. and Sakaguchi, G. (1977) Botulogenic properties of vegetables with special reference to the molecular size of the toxin in them. J. Food Safety 1, 53-65. Sugiyama, H. (1980) CIostridiurn botulinurn neurotoxin. Microbiol. Rev. 44, 419-448. Syoto, B. and Kubo, S. (1981) Separation and characterization of heavy and light chains of CIostridium botulinum type C toxin and their reconstitution. J. Biol. Chem. 256. 3712 3717. Takamizawa, K., lwamori, M., Kozaki, S., Sakaguchi, G., Tanaka, R., Takayama, H. and Nagai, Y. (1986) TLC immunostaining characterization of Clostridium botulinum type A neurotoxin binding to gangliosides and free fatty acids. FEBS Lett. 201, 229 232. Weickert, M.J., Chambliss, G.H. and Sugiyama, H. (1986) Production of toxin by CIostridium hotulinum type A strains cured of plasmids. Appl. Environ. Microbiol. 51, 52 56. Williams, R.S., Tse, C.K., Dolly, J.O., Hambleton, P. and Melling, J. (1983) Radioiodination of botulinum neurotoxin type A with retention of biological activity and its binding to brain synaptosomes. Eur. J. Biochem. 131, 437-445.
124 Woodburn, M.J., Somers, E., Rodriguez, J. and Schantz, E.J. (1979) Heat inactivation rates of botulinum toxins A, B, E and F in some foods and buffers, J. Food Sci. 44, 1658-1661. Woolford, A.L., Schantz, E.J. and Woodburn, M.J. (1978) Heat inactivation of botulinum toxin type A in some convenience foods after frozen storage. J. Food Sci. 43, 622-624. Yokosawa, N., Tsuzuki, K., Syuto, B. and Oguma, K. (1986) Activation of Clostridium botulinum type E toxin purified by two different procedures. J. Gen. Microbiol. 132, 1981-1988.
113
F O O D 80007
Clostridium botulinum toxins A.H.W.
Hauschild
Microbiology Research Division, Health Protection Branch, Ottawa, Ontario, Canada
Structure, activation, destruction, lethality, genesis, neurotoxic action and medical application of botulinal toxins are reviewed. Key words: Clostridium botulinum; Toxins; Foods
Introduction Eight distinct toxins of Clostridium botulinum designated A, B, C 1, C z, D, E, F and G, are presently recognized. They are all proteins made up of about 20 c o m m o n amino acids and, with the exception of toxin C z, exhibit a characteristic neurotoxic action. Toxins are formed by each of the four diverse groups of C. botulinum. In addition, recent isolates from cases of infant botulism, identified as C. butyricum and C. barati, were found to produce botulinal toxins E (Aureli et al., 1986) and F (Hall et al., 1985), respectively (Table I). In general, individual strains of C. botulinum produce a single toxin. Exceptions are some rare isolates of group I, each producing one major and one minor toxin (Gim6nez and Ciccarelli, 1978; Hatheway et al., 1981; McCroskey and Hatheway,
TABLE I
Clostridia producing botulinal toxins Producer species
Group
Toxin type
C. botulinum
1 II III IV
A, B, F B,E,F C I , C 2, D G
C. butyricum
E
C. barati
F
Correspondence address: Microbiology Research Division; Health Protection Branch, Ottawa, Ontario, Canada K 1 A 0 L 2 . 0 1 6 8 - 1 6 0 5 / 9 0 / $ 0 3 . 5 0 ~ 1990 Elsevier Science Publishers B.V.
114 T A B L E 11 T o x i n s of CIostridium botulinum
C. botulinum
T o x i n type
Group
Type
1
A
Subtype
Major
Minor
AB AF
A A A
B F
B
F
B B B F
II
B E F
B E F
-
I11
C
(71
C 2, D C2 C t, C~
BA BF
IV
C~ C/~
D
D
G
G
A F
1984; Poumeyrol et al., 1983), and strains of types C and D which may form up to three toxins each (Eklund and Poysky, 1981) (Table II). The type designation of strains is identical to that of their major toxin. Toxins and antitoxins of types B and F each cross-neutralize completely between groups I and II, but the type-specific toxins of the two groups are not identic'a_l (Shimizu et al., 1974). Rymkiewicz et al. (1979) reported antitoxin titers for sera to group II toxin B that were six times higher in titrations of homologous toxin than in titrations of group I toxin B. Toxins C and D and their antitoxins also cross-react (Oguma et al., 1980), and antitoxin E is capable of neutralizing small quantities of toxin F (Eklund et al., 1967). Botulinal toxins are produced during growth, but little toxin is released from the intact cell. The main release of toxin starts at the end of the growth phase and coincides with cell lysis (Duda and Slack, 1969; Siegel and Metzger, 1979).
Structure The botulinal toxins may exist in four molecular sizes of approximately 7S, 12S, 16S and 19S, designated S (small), M (medium), L (large) and LL (extra large) toxins, respectively (Sakaguchi et al., 1981). The molecular weights range from 150 to 900 kDa (DasGupta, 1981; Sugiyama, 1980). Only the M(12S) and L(16S) forms are encountered in toxic foods and cultures (Hauschild and Hilsheimer, 1969; Sugii and Sakaguchi, 1977). Of these, the M form is the more c o m m o n natural toxin. It is an aggregate of a toxic component, the small (7S) single-chain toxin, with an atoxic
115
component. The L toxin also includes a haemagglutinin in the complex. Natural toxins (M and L) are commonly referred to as 'progenitor toxins', and S toxin is referred to as 'derivative toxin' (Lamanna and Sakaguchi, 1971). LL toxin was the first botulinal toxin to be purified and crystallized (Lamanna and Carr, 1967) and is only known for type A. This toxin has not been encountered in cultures and is likely an artificial aggregate. Under mild alkaline conditions, progenitor toxins dissociate into S toxin and atoxic components which can be separated by ion exchange chromatography (Boroff and DasGupta, 1971). The derivative (S) toxin, a single-chain protein, is 'nicked' by a number of proteases, including proteases of group I C. botulinum, into a dichain molecule, with the two chains remaining associated by a disulfide bond. This bond may be severed by reducing agents, e.g., dithiothreitol. Only toxin C 2 has no covalent linkage between the two chains. The two components of nicked toxin, a light (L) and heavy (H) chain, have molecular weights of roughly 50 and 100 kDa, respectively, and can be separated by polyacrylamide gel electrophoresis (Sugiyama, 1980). Individually, the two components are nontoxic, but toxicity can be restored by reestablishing the covalent bond through oxidation. Toxin C 2 activity can be restored by simply mixing the two chains (Ohishi et al., 1980). The L and H chains are antigenically distinct, and antibodies to either of the two, particularly the H component, may neutralize the complete toxin (Kozaki et al., 1986; Syoto and Kubo, 1981). Toxins A, B~ C 1, C 2, D, E and F have been purified and characterized. They all have basically the same structure, with molecular weights of the S toxin, L and H chains in the order of 150, 50 and 100 kDa, respectively. Estimates based on amino acid composition were essentially the same ( D a s G u p t a and Woody, 1984).
Activation
The lethality of progenitor and derivative toxins can be increased by a few log10 units in the presence of proteolytic enzymes. Group II toxins (B, E and F) and toxins C 2 and G are particularly susceptible to activation (Eklund and Poysky, 1972; Iida, 1968; Solomon et al., 1985). Yokosawa et al. (1986) reported 30-fold higher activation of toxin E extracted from cells as compared to the toxin in culture fluid, which indicates that some activation takes place also in 'non-proteolytic' group II cultures. Trypsin activation of botulinal toxin is accompanied by conversion of single-chain toxin to nicked toxin. However, the process of nicking seems irrelevant to the mechanism of activation because activation with trypsin at a low pH (4.5) or with a 'trypsin-like enzyme' (TLE) from a proteolytic type B culture has been shown to proceed without a significant degree of nicking ( D a s G u p t a and Sugiyama, 1976; Ohishi and Sakaguchi, 1977). The relevant cleavage associated with activation occurs on the H chain (Ohishi et al., 1980) and likely involves the hydrolysis of a lysyl bond (Miura, 1974). The cleavage site remains obscure. D a s G u p t a and Sugiyama (1976) found no detectable
116
change in molecular structure as a result of toxin E activation with TLE, while Ohishi (1987) reported a molecular weight change from 101 kDa to 88 kDa in the trypsin-activated H (II) component of toxin C 2.
Inactivation
Two aspects of toxin inactivation are of particular interest: the thermal destruction of toxins in foods, and their destruction in drinking water at ambient temperatures. Thermal inactivation of botulinal toxins is nonlinear. The destruction curves are generally diphasic, with an initial steep decline levelling off with time (Cartwright and Lauffer, 1958; Woodburn et al., 1979). The curves may be explained by differences in the thermostability of progenitor toxin, variation in the degree of hydrogen bonding, or aggregation during heating (Lamanna, 1959; Licciardello et al., 1967b). The nonlinear heat inactivation curves preclude the expression of thermoresistance in D-values. Instead, the heat resistance of botulinal toxins is expressed as the time required for a given toxin concentration (Co) to be reduced to a single LDs0 dose ( F value). With Co of about 1 0 4 m o u s e LDs0/g, a level often encountered in toxic foods, F values for toxins A, B, E, F and G in a variety of foods at 78-80 ° C varied from < 1 min to 2 min (Bradshaw et al., 1979, 1981; Licciardello, 1967a). With Co of about 105 LDs0/g, F values at 79 o C ranged from 2 to 20 min for toxin A, from 1.5 to 10 min for toxin B and from 1.5 to 5 rain for toxin E (Woodburn et al., 1979). Relatively high F values have been recorded in acid foods such as tomato juice. This is consistent with maximum thermoresistance of botulinal toxins at pH 4-5. F values are increased also in the presence of proteins and colloidal materials and by increased levels of ionic strength. Consequently, foods are generally more protective than buffers (Bradshaw et al., 1979, 1981; Woodburn et al., 1979; Woolford et al., 1978). For safe thermal inactivation of botulinal toxins at levels up to 10 s LDs0/g in foods, Woodburn et al. (1979) recommended time/temperature combinations of 20 min at 7 0 ° C or 5 min at 85 °C. In contrast to the nonlinear inactivation of toxin with time, F values plotted against temperature follow a straight line. Their slopes indicate Z-values of 3-5 ° C for toxins A and B, 4 - 6 ° C for toxins F and G, and 7 8 ° C for toxin E (Bradshaw et ai., 1979, 1981; Licciardello, 1967a). In spite of its significance, the question of toxin stability in drinking water has rarely been addressed experimentally. Toxin inactivation in water is nonlinear and, like heat destruction, slows down with time. Schantz (personal communication) observed a 2.5 log10 decrease of toxin A in drinking water after 2 days, but a total decrease of only 3.5 log m units after 6 days. Relative toxin stability in water has also been reported by others (Brygoo, 1953; Markarjan et al., 1960). For rapid detoxification of drinking water within 1 h, about 10 mg of residual chlorine per 1
117
would be required. Alternative means of toxin removal involve alum treatment and filtration, or active charcoal treatment.
Lethality When botulinal toxins are administered by the parenteral route, the atoxic component has little or no effect on toxicity (Sakaguchi et al., 1984). In contrast, oral toxicities increase by up to 1 0 4 log10 units with increasing molecular weight of the toxin indicating that the atoxic moiety protects the toxic component in the digestive tract (Ohishi et al., 1977; Sakaguchi et al., 1981). Protection of progenitor toxin from gastric juice or pepsin by its atoxic component has been demonstrated in vitro (Sakaguchi et al., 1981). The protective role of the atoxic component ends with the toxin uptake into the lymph which occurs predominantly in the duodenum and jejunum (Ohishi, 1983a). Before entering the lymph, the atoxic portion is shed, and only the 7 S form is encountered in the lymph and blood stream (Bonventre, 1979; Hildebrand et al., 1961). Lethal doses of ingested toxin for humans are estimated as the toxin content of the food consumed by fatal or near-fatal cases of botulism without effective treatment. Because of uncertainties about the amount of toxin ingested and variations in the proportions of L and M toxins among toxic foods, such estimates can only be approximate. These range from about 5 × 10 3 tO 1 × 105 mouse (i.p.) LDs0 for group I toxins A and B (Bonventre, 1979; Meyer and Eddie, 1951; Morton, 1961) and from 1 ×105 to 5 × 1 0 s mouse LDs0 for group II toxins B and E (Dolman et al., 1955; Ralovich and Barna, 1966). The lethality of toxin F in humans is likely much lower. In monkeys, the oral lethal dose of group I toxin F was 5 × 104 mouse LDs0/kg (Dolman and Murakami, 1961) which is comparable to the lethality of toxin G (Ciccaretli et al., 1977) but more than a log m unit lower than that of toxins A, B and E (0.5-2.5 x 103 mouse LDs0/kg ) (Smith, 1977; Dolman and Murakami, 1961).
Genetics of toxigenesis A number of bacterial protein toxins are specified by plasmids or bacteriophages (Murphy et al., 1984; H~irtlein et al., 1984). Plasmids and bacteriophages have been demonstrated in nearly all types of C. botulinum, but no association between botulinal toxigenesis and plasmids has been found (Strom et al., 1984; Weickert et al., 1986), and mediation of toxin production by bacteriophages has been established only for toxins C 1 and D of C. botulinum types C and D. The bacteriophages of these types are characterized by hexagonal heads and long, partially sheathed tails of up to 450 nm (Eklund et al., 1972; Inoue and Iida, 1968). Bacteriophages of other C. botulinum types show similar structures.
118 Loss of bacteriophages by toxigenic strains of types C and D, e.g., by repeated transfer, ultraviolet irradiation or acridine orange treatment, is accompanied by the loss of ability to produce C~ and D toxins, and loss of resistance to lysis by bacteriophages of the toxic parent strain or related phages. This process is reversed when nontoxigenic cultures are reinfected with specific bacteriophages. Both toxins of a given strain are controlled by a single phage (Eklund and Poysky, 1981; Inoue and Iida, 1970). Some strains of types C and D may be converted from one type to another by curing them of their prophages and reinfecting them with viruses from types D and C, respectively (Eklund and Poysky, 1974, 1981). Eklund et al. (1974) also demonstrated compatibility between strains and bacteriophages of different species. A strain of type C, cured of its prophage and reinfected with phage NA1 of Clostridiurn novyi type A, also gained the ability to produce C. novyi alpha toxin. These conversions are all reversible. Control of toxigenesis by bacteriophages has not been established for other types of C. botulinum. In case of a stable host-phage relationship, such a control might be difficult to demonstrate. In strains of types C and D, this relationship is generally unstable as indicated by the loss of phages after transfers. This loss is even accelerated when antiserum against the host-specific phage is added to growing cultures to avoid reinfection of phage-cured cells (Eklund and Poysky, 1981: Iida et al., 1981).
Neurotoxic action
With the exception of toxin C 2, the botulinal toxins exhibit a specific neurotoxic action which results in the blockage of transmitter release at the neuromuscular junctions. Both cholinergic and adrenergic systems are affected, but much higher concentrations are required to inhibit the release of noradrenaline than that of acetyl choline (Habermann, 1981; Simpson, 1979). Three stages in the neurotoxic mechanism have been recognized: toxin binding, toxin internalization and blockage of transmitter release (Black and Dolly, 1986b; Simpson, 1981a). The first step involves the interaction of toxin with specific receptor sites on the presynaptic membrane which is mediated by the H chain (Bandyopadhyay et al., 1987; Black and Dolly, 1986a, 1986b; Kitamura, 1976). The receptor likely contains sialic acid as an essential ingredient because neuraminidase and gangliosides both interfere with toxin binding (Bigalke et al., 1986; Williams et al., 1983), and gangliosides are potent toxin receptors (Ochanda et al., 1986; Takamizawa et al., 1986). The binding sites are not the same for each toxin, e.g., binding of toxin A to synaptosomes is in competition with toxin E but not with toxin B (Kozaki, 1979). The second step involves translocation of the toxin, or a fraction of it, through the presynaptic membrane (Black and Dolly, 1986a; Donovan and Middlebrook, 1986). The process only occurs after binding to the receptor and is energy (ATP) and temperature dependent (Black and Dolly, 1986b; Simpson, 1981b). From here on, the toxin can no longer be neutralized (Simpson, 1981a).
119 The final stage involves the blockage of neurotransmitter release. Since diffeeent transmitters are affected, the action site is likely the same for all transmitter systems (Bigalke et al., 1985). Pumplin and Reese (1977) demonstrated interference of botulinal toxin in the fusion of acetyl choline containing cytoplasmic vesicles with the presynaptic membrane. It has long been postulated that the underlying toxin reaction is enzymatic (Simpson, 1981b). Ohashi and Narumiya (1987a) and Ohashi et al. (1987b) reported enzymatic A D P ribosyltransferase activity of C1 and D toxins involving a membrane protein with a molecular weight of 21 kDa: N A D + protein (21 kDa) ~ ADP-ribose-protein + nicotinamide. The authors suggested that the neurotoxins may catalyze the transfer of ADPribose from N A D o a specific membrane protein involved in the exocytosis of transmitter (Ohashi and Narumiya, 1987a). Matsuoka et al. (1987) confirmed the enzymatic activity of toxins C1 and D, except that the protein substrates had slightly larger molecular weights (24-26 kDa). Aktories et al. (1987) reported ribosyl transferase activity of C. botulinum type C with a 21-kDa protein as substrate, but the activity was associated with a low-molecular weight (24 kDa) enzyme which could not be toxin C 1. No enzymic activity has been demonstrated for any of the other neurotoxins.
Action of toxin C 2
Toxin C 2 differs from other botulinal toxins in the absence of a covalent bond between L and H chains, its lack of neurotoxicity, and a unique pathological mechanism manifesting itself in the leakage of plasma protein into the intestinal lumen, intestinal fluid accumulation, vacuolization of epithelial cells, intercellular edema, necrosis of the mucosa, increased vascular permeability, hypotension, and hemorrhaging of the lungs (Aktories et al., 1986a; Ohishi and Ogadiri, 1984; Simpson, 1982, 1984). Toxin C 2 ribosylates a specific protein of 43 kDa which was identified as G-actin (Aktories et al., 1986a, 1986b; Ohishi, 1986). As a consequence of actin binding, the microfilament network structure of the host cell becomes disorganized (Reuner et al., 1987). The ribosyltransferase activity resides in the L chain, whereas the specific binding is associated with the H chain (Aktories, 1986a; Ohishi, 1983b, 1986).
Beneficial effects of botulinal toxins
This review would be incomplete without the mention of some medical applications of botulinal toxin. Small doses of toxin A in the order of 100 mouse M L D have been used for several years in the treatment of strabismus as an alternative to surgery. Injection of excessively pulling muscles with toxip ~auses temporary local
120
paralysis and results in long-lasting muscle realignments (Magoon and Scott, 1987: Schantz and Scott, 1981). Similarly, eyelid spasms (Cohen et al., 1986) and closures (Katz and Rosenberg, 1987), spasmodic dysphonia (Miller et al., 1987) and urethal sphincter spasticity (Dykstra et al., 1986) have been treated successfully, though with transient relief.
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124 Woodburn, M.J., Somers, E., Rodriguez, J. and Schantz, E.J. (1979) Heat inactivation rates of botulinum toxins A, B, E and F in some foods and buffers, J. Food Sci. 44, 1658-1661. Woolford, A.L., Schantz, E.J. and Woodburn, M.J. (1978) Heat inactivation of botulinum toxin type A in some convenience foods after frozen storage. J. Food Sci. 43, 622-624. Yokosawa, N., Tsuzuki, K., Syuto, B. and Oguma, K. (1986) Activation of Clostridium botulinum type E toxin purified by two different procedures. J. Gen. Microbiol. 132, 1981-1988.
